Test strip with slot vent opening

ABSTRACT

A test strip with a covering layer having a novel slot. The slot divides the inventive covering layer into two parts and provides a vent opening that allows air to escape as fluid enters a cavity or sample receiving chamber formed in the test strip. In preferred embodiments, the covering layer is clear such that the user can see through it and the slot doubles as a “fill line.” The user can thus watch the fluid sample enter the test strip, progress through the capillary cavity, and then stop at the slot or fill-line. This provides positive assurance to the user that the sample size is sufficient and the test strip has been filled properly. The present invention also provides an advantageous method of mass-producing the inventive test strips without having to align the slot or vent opening laterally with respect to the test strips and without having to punch a vent opening. The method is also well suited to mass production by roll processing techniques.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 60/480,397, filed Jun. 20, 2003.

FIELD OF THE INVENTION

The present invention relates generally to the testing of body fluidsfor concentration of analytes and more particularly to a test strip orbiosensor for such testing.

BACKGROUND

Test strips are often used to measure the presence and/or concentrationsof selected analytes in test samples. For example, a variety of teststrips are used to measure glucose concentrations in blood to monitorthe blood sugar level of people with diabetes. These test strips includea reaction chamber into which a reagent composition has been deposited.Current trends in test strips require smaller test samples and fasteranalysis times. This provides a significant benefit to the patient,allowing the use of smaller blood samples that can be obtained from lesssensitive areas of the body. Additionally, faster test times and moreaccurate results enable patients to better control their blood sugarlevel. In connection with smaller sample volumes, it is known to providetest strips having a sufficiently small reaction chamber such thatsample fluid is drawn therein by capillary action, which is a phenomenonresulting from the surface tension of the sample fluid and thethermodynamic tendency of a liquid to minimize its surface area. Forexample, U.S. Pat. No. 5,141,868 discloses a test strip having a cavitysized sufficiently small to draw sample liquid therein by capillaryaction. The cavity is defined by two parallel plates spaced about 1 mmapart by two epoxy strips extending lengthwise along lateral sides ofthe plates. The cavity is open at both ends, one of which receives thesample, and the other of which allows air to escape. The cavity includesan electrode structure and carries a coating of a material appropriateto the test to be performed by the test strip.

Various other test strip designs include capillary cavities that drawsample fluid therein and include vent openings to allow air to escape.Typically, the vent opening is punched or otherwise formed in either thetop or bottom film that forms the sample receiving cavity. Manufacturingissues arise because of the need to precisely locate the vent holerelative to the cavity. For example, if the cavity is centrally disposedlengthwise within the test strip, a vent hole aligned left or right ofcenter may not connect or communicate with the cavity. Since the stripsare typically mass-produced from a continuous web, an error in alignmentof the vent hole can affect hundreds or even thousands of test strips.

Moreover, punching a hole for the vent opening requires a separateprocess step and a cutting die or other equipment to form the opening.In view of cavity sizes becoming increasingly smaller in modern teststrips, forming the vent opening has become a more delicate processstep. It would be desirable to reduce the potential for error and toreduce the costs associated with forming the vent opening in test stripsrequiring the same.

SUMMARY OF THE INVENTION

The present invention provides a test strip with a covering layer havinga novel slot. The slot divides the covering layer into two parts andprovides a vent opening that allows air to escape a cavity or samplereceiving chamber formed in the test strip as fluid enters it. Inpreferred embodiments, the covering layer is clear, such that the usercan see through it and the slot doubles as a “fill line.” The user canthus watch the fluid sample enter the test strip, progress through thecapillary cavity, and then stop at the slot or fill-line. This providespositive assurance to the user that the sample size is sufficient andthe test strip has been filled properly. Advantageously, the inventivetest strips can be mass-produced without having to align the slotlaterally with respect to the test strip and without having to punch avent opening.

In one form thereof, the present invention provides a test stripcomprising a covering layer overlying a base substrate. The basesubstrate has a reagent layer disposed on it that reacts with the fluidsample and produces a measurable response that can be correlated to theconcentration of the analyte being measured. The covering layer includesa chamber cover and a body cover having a slot therebetween. The slot ispositioned over the reagent layer. A sample receiving chamber isdisposed between the base substrate and the covering layer, and the slotcommunicates with the sample receiving chamber. The slot defines a ventopening in the covering layer that allows air to escape as fluid entersthe sample receiving chamber.

Preferably, the slot comprises a gap which forms a space between thebody cover and the chamber cover, although the covering layer can be ofunitary construction, with the slot forming a recess or groove in thebottom thereof. The slot can also be formed by having one of the chambercover and body cover overlap the other. In all cases, the slot ispreferably straight and extends across the width of the covering layer,oriented substantially perpendicular to the lengthwise or longitudinalaxis of the test strip. This configuration of the slot providesadvantages in mass-producing the test strips, as described below.

In another preferred form, the test strip includes a spacing layerdisposed between the covering layer and the base substrate. The spacinglayer includes a void that further defines the height, perimeter andlength of the sample receiving chamber between the base substrate andthe covering layer. That is, the sample receiving chamber is bounded onits sides by vertical walls created by the void, on its top by thecovering layer, and on its bottom by a reagent layer that preferablyoverlies the base substrate. The void is shaped as an elongate channelthat begins at a fluid receiving opening at an edge of the test strip,extends along the lengthwise direction of the strip, and terminates at alocation that is substantially aligned with the vent opening or slot.

The chamber cover is preferably sized to overlie substantially theentire length of the sample receiving chamber, whose length isestablished by the length of the void in the spacing layer, as justdiscussed. The interior end of the chamber cover, which corresponds tothe location of the slot, is substantially aligned with the interior endof the sample receiving chamber. In this configuration, the air spacedefined by the slot and the air space occupied by the interior end ofthe sample receiving chamber connect, or overlap, such that the samplereceiving chamber is in communication with the slot or vent opening, anda means for air to escape the sample receiving chamber is provided.Thus, the sample receiving chamber communicates with ambient air fromthe fluid receiving opening at one end and from the vent opening at itsother end. The small size of the sample receiving chamber produces acapillary effect that quickly draws fluid sample therein, displacing airthrough the vent opening. In preferred embodiments, the slot is formedas a gap and at least a portion of the air displaced exits from the topof the test strip.

Electrodes are preferably formed on the base substrate and are disposedin the sample receiving chamber. As noted above, a reagent layer isdisposed in the sample receiving chamber and covers at least one, andpreferably both, electrodes. More preferably, the reagent layer extendsunder the spacing layer and is actually sandwiched between the spacinglayer and the base substrate, extending to the lateral edges of the teststrip. The reagent layer thus defines most or all of the bottom surfaceof the sample receiving chamber. This reagent stripe configurationprovides advantages in manufacturing, as described in further detailbelow.

Another preferred aspect of the inventive test strips involves thechamber cover being transparent or translucent above the samplereceiving chamber. Fluid entering the sample receiving chamber is thusvisible through the chamber cover. Further, before the test strip hasbeen used, the sample receiving chamber is empty and the bottom thereofis visible through the chamber cover. If the inventive test strips areused for testing blood as the fluid sample, for example, it is desirableto color the reagent layer a color that contrasts the red color ofblood.

The spacing layer is preferably formed of an opaque color that contrastswith both the color of the fluid sample and that of the reagent layer.Thus, when viewed from the top of the test strip, the user sees throughthe transparent chamber cover and sees the color of the floor of thesample receiving chamber bounded by the contrasting color of the spacinglayer. Alternatively, the contrasting color may be provided, e.g., byprinting on the transparent chamber cover. A blood sample is depositedat the fluid receiving opening at the edge of the strip and is quicklydrawn into the sample receiving chamber. The user can easily watch thered colored blood moving into the sample receiving chamber against thecontrasting background, which provides a positive indication to the userthat a sufficient size sample of blood was provided.

In another preferred aspect of the present invention, at least theunderside of the chamber cover is hydrophilic, which promotes quickwicking of the sample into the elongated chamber at least as far as thevent opening. By contrast, the body cover is hydrophobic, and since thebody cover defines an edge of the slot, it prevents fluid sample fromwicking beyond the slot or vent opening. These contrasting hydrophobicand hydrophilic properties result in the sample fluid being quicklydrawn into the sample receiving chamber, yet fluid movement is quicklyhalted when the sample reaches the area in the chamber that is alignedwith the slot. When the slot is substantially straight, the sample formsa corresponding straight terminal edge aligned therewith. When combinedwith the transparent chamber cover and other preferred features notedabove, the user is thus provided with a clearly defined and visible“fill line” corresponding to the slot. The user can watch the fluidsample quickly enter the sample receiving chamber and then stop at thefill line, confirming that the sample size was sufficient and that thetest strip was filled properly.

In another preferred form, contact pads are formed on the base substrateat a meter insertion end of the test strip and electrode traces extendalong the base substrate and connect the electrodes to the contact pads.The covering and/or spacer layer described above preferably covers mostof the length of the test strip, but terminates short of the meterinsertion end, thereby exposing the contact pads at the meter insertionend of the strip. This allows the contact pads to mate withcorresponding electrical connections in a meter that reads the teststrips.

In another form thereof, the present invention provides a method of massproducing the novel test strips described above. In this inventivemethod, a web of base substrate material is provided. A plurality ofelectrode sets is formed on the web. In preferred embodiments, theelectrode sets are formed by laser ablation, more preferably, by broadfield laser ablation. A series of cavities is also formed in the web. Ina preferred embodiment, the cavities are formed by providing acontinuous web of spacing layer material having the shape of thecavities cut out and spaced equidistantly. Each one of the cavities isaligned with a respective one of the electrode sets.

A reagent layer is provided and covers at least one electrode of eachelectrode set. In a preferred form, the reagent layer is applied to theweb before the cavities are formed, such that the reagent layer can beapplied in a continuous “stripe” of uniform thickness. Finally, acovering layer preferably made from two pieces is placed over andlaminated to the web such that the two pieces are separated by a gap andthe gap is positioned over the series of cavities. Preferably, bothpieces of the covering layer are applied at the same time. The web isthen cut into the plurality of test strips.

As noted above, this mass production method avoids the need to align thevent opening laterally relative to the test strips. Moreover, theinventive method is further advantageous because it avoids the need tootherwise form an aperture in the covering layer or base layer. Themethod is also well-suited to mass production of the test strips by rollprocessing techniques, as described herein.

In one form thereof, the present invention provides a test stripcomprising a covering layer overlying a base substrate. The basesubstrate has a reagent layer disposed on it. The covering layerincludes a chamber cover and a body cover having a slot therebetween.The body cover is thicker than the chamber cover. A sample receivingchamber is disposed between the base substrate and the covering layer,and the slot communicates with the sample receiving chamber. The slotdefines a vent opening in the covering layer that allows air to escapeas fluid enters the sample receiving chamber.

Advantageously, a thicker body cover absorbs more of the pressure orforce imparted to the web as the assembly is rewound and stored duringprocessing. Thus, if any adhesive squeezes out of the web as it isrewound, the adhesive will typically squeeze out around the body coverand not the chamber cover. This reduces the possibility of the adhesivesqueezing out from under the chamber cover during roll processing andentering the capillary zone where it could degrade or destroy the teststrips ultimately produced.

The present invention provides a very easy-to-dose test strip andprovides a robust but flexible manufacturing process. The various otherfeatures that characterize the invention are pointed out withparticularity in the attached claims. For a better understanding of theinvention, its advantages, and objectives obtained therefrom, referenceshould be made to the drawings and to the accompanying description, inwhich there is illustrated and described preferred embodiments of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

Referring now to the drawings, wherein like reference numerals andletters indicate corresponding structure throughout the several views:

FIG. 1 is a perspective view of a test strip or biosensor in accordancewith the present invention.

FIG. 1A is an enlarged fragmentary perspective view of the test stripshown in FIG. 1, illustrating one embodiment of the novel vent openingor slot.

FIG. 1B is an enlarged fragmentary perspective view illustrating analternate embodiment of the vent opening or slot in accordance with thepresent invention.

FIG. 1C is an enlarged fragmentary perspective view illustrating anotheralternate embodiment of the vent opening or slot and also illustratingan alternate configuration of the opening to the sample receivingchamber of the biosensor in accordance with the present invention.

FIG. 2 is an exploded, perspective view of the biosensor of FIG. 1.

FIG. 3 is a cross-sectional view of a portion of the biosensor of FIG.1, additionally illustrating adhesive layers that have been omitted fromFIGS. 1-2.

FIG. 4 is a top, plan view of a portion of the biosensor of FIG. 1, withportions broken away to show underlying details.

FIGS. 5 and 5A show a process flow diagram for a method for producing abiosensor in accordance with the present invention.

FIG. 6 is a perspective view showing the reel-to-reel processing andcutting of a web material useful in forming the bottom substrate of thebiosensor of the present invention.

FIG. 7 is a perspective view of a portion of a webbing, showing anexemplary pattern of electrical components on the base substrate.

FIG. 8 is a perspective view of a portion of the webbing of FIG. 7 andincluding a reagent composition coated thereon.

FIG. 9 is an exploded, perspective view showing a spacing layer and theassociated adhesive layers and release liners.

FIG. 10 is an exploded perspective view of a portion of the spacinglayer with pre-capillary chambers cut out and the spacing layer beingaligned for lamination to a base substrate having electrode patternsthereon.

FIG. 11 is a perspective view of an assembly of the base substrate withthe spacing layer.

FIG. 12 is an exploded, perspective view showing the combination of thebody and chamber covers for assembly onto the base substrate and spacinglayer.

FIG. 13 is a perspective view of a portion of an assembly including theseveral layers comprising the biosensor.

FIG. 14 is a perspective view of a portion of webbing including severaldetachable biosensors.

FIG. 15 is a perspective view of a single biosensor separated from theassembled webbing.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

For the purposes of promoting an understanding of the principles of theinvention, reference will now be made to the specific embodimentsillustrated herein and specific language will be used to describe thesame. It will nevertheless be understood that no limitation of the scopeof the invention is thereby intended. Any alterations and furthermodifications in the described processes or devices, and any furtherapplications of the principles of the invention as described herein, arecontemplated as would normally occur to one skilled in the art to whichthe invention relates.

System

The present invention relates to a system that is useful for assessingan analyte in a sample fluid. The system includes devices and methodsfor evaluating the sample fluid for the target analyte. As more fullydescribed hereafter, the evaluation may range from detecting thepresence of the analyte to determining the concentration of the analyte.The analyte and the sample fluid may be any for which the test system isappropriate. For purposes of explanation only, a preferred embodiment isdescribed in which the analyte is glucose and the sample fluid is bloodor interstitial fluid. However, the present invention clearly is not solimited in scope.

Sensor

One component of the system is an electrochemical sensor including asample-receiving chamber for the sample fluid, and a suitable reagentfor producing an electrochemical signal in the presence of the testanalyte. The sensor preferably comprises a disposable test strip,particularly one having a laminar construction providing an edge openingwhich communicates with the sample-receiving chamber. The reagent isdisposed within the sample-receiving chamber in position to provide theelectrochemical signal to a working electrode also positioned within thechamber. In appropriate circumstances, such as for glucose detection,the reagent may contain an enzyme and optionally a mediator.

Meter

The sensor is used in combination with a meter for determination of theanalyte in the sample fluid. The meter conventionally includes aconnection with the electrodes of the sensor and circuitry to evaluatethe electrochemical signal corresponding to the concentration of theanalyte. The meter may also include means for determining that thesample fluid has been received by the sensor, and that the amount ofsample fluid is sufficient for testing. The meter typically will storeand display the results of the analysis, or may alternatively providethe data to a separate device.

Analyte—Characteristic

The system can provide either a qualitative or quantitative indicationfor the analyte. In one embodiment, the system indicates simply thepresence of the analyte in the sample fluid. The system may also providea reading of the quantity or concentration of the analyte in the samplefluid. In a preferred embodiment, it is a feature of the presentinvention that a highly accurate and precise reading of the analyteconcentration is quickly obtained from a small volume of sample fluid.

Analyte—Type

The system is useful for the determination of a wide variety ofanalytes. The test strip, for example, is readily adapted for use withany suitable chemistry that can be used to assess the presence of theanalyte. Most preferably, the system is configured and used for thetesting of an analyte in a biological fluid. Such analytes may include,for example, glucose, cholesterol, HDL cholesterol, triglycerides,lactates, lactate dehydrogenase, alcohol, uric acid, and 3-hydroxybutricacid (ketone bodies). Commensurate modifications to the system will beapparent to those skilled in the art. For purposes of explanation, andin a particularly preferred embodiment, the system is described withrespect to the detection of glucose in a biological fluid.

Interferants

Test methodologies may be variously affected by the presence ofinterferants in the sample fluid. For example, the testing for glucosein a blood sample may be impacted by such factors as oxygen, bilirubin,hematocrit, uric acid, ascorbate, acetaminophen, galactose, maltose, andlipids. The present system is adaptable to minimize or eliminate theadverse effects of interferants that may also be present in the samplefluid. These effects may be addressed by appropriate selection of testmaterials and parameters, such as by the selection of chemistries thatare known to be impacted less, or not at all, by possible interferants.As is known in the art, other steps may also be taken to addresspossible interferant effects, such as the use of coatings or films thatprevent the interferant from entering the test zone. In addition,modifications to the electrode configurations or interrogation methodscan be used to minimize the effect of interferants.

Fluid Type

The system is useful with a wide variety of sample fluids, and ispreferably used for the detection of analytes in a biological fluid. Inthis context, the term “biological fluid” includes any bodily fluid inwhich the analyte can be measured, for example, interstitial fluid,dermal fluid, sweat, tears, urine, amniotic fluid, spinal fluid andblood. The term “blood” in the context of the invention includes wholeblood and its cell-free components, namely plasma and serum. Inaddition, the system is useful in connection with control fluids thatare used in conventional fashion to verify the integrity of the systemfor testing.

In a preferred embodiment, the system is employed for the testing ofglucose. The sample fluid in this instance may specifically include, forexample, fresh capillary blood obtained from the finger tip or approvedalternate sites (e.g., forearm, palm, ear lobe, upper arm, calf andthigh), fresh venous blood, and control solutions supplied with or forthe system.

The fluid may be acquired and delivered to the test strip in anyfashion. For example, a blood sample may be obtained in conventionalfashion by incising the skin, such as with a lancet, and then contactingthe test strip with fluid that appears at the skin surface. It is anaspect of the present invention that the test strip is useful with verysmall fluid samples. It is therefore a desirable feature of theinvention that only a slight incising of the skin is necessary toproduce the volume of fluid required for the test, and the pain andother concerns with such method can be minimized or eliminated.

It is also well known that different locations on the skin will producemore or less amounts of blood upon lancing. The finger tip, for example,is a commonly used site for obtaining a blood sample because it producesa relatively large amount of blood upon lancing. However, it is alsoknown that areas that produce larger volumes of blood are generallyassociated with greater degrees of pain for the user. It is therefore anadditional advantage of the present system that the required volume ofsample fluid is sufficiently small that the test strip is useful withthe amount of blood typically obtained upon lancing less productive, butalso less painful, areas of the skin, such as the palm and upper arm.The use of these locations to obtain sample fluids for testing issometimes referred to as “alternate site testing”. The present inventionis particularly well suited to use with sample fluids, e.g., blood orinterstitial fluid, obtained at these alternate sites.

Test Strip—General

Introduction.

The test strip includes several basic components. The strip comprises asmall body defining a chamber in which the sample fluid is received fortesting. This “sample-receiving chamber” is filled with the sample fluidby suitable means, preferably by capillary action, but also optionallyassisted by pressure or vacuum. The sample-receiving chamber includeselectrodes and chemistry suitable for producing an electrochemicalsignal indicative of the analyte in the sample fluid. Basic Description.

Referring in particular to the drawings, there is shown a preferredembodiment of a test strip useful in accordance with the presentinvention. The test strip 10 includes a base substrate 12, a spacinglayer 14 and a covering layer 16 comprising body cover 18 and chambercover 20. The spacing layer 14 includes a void portion 22 to provide asample-receiving chamber 24 extending between the base substrate 12 andthe covering layer 16.

The base substrate 12 carries an electrode system 26 including aplurality of electrodes 28 and electrode traces 30 terminating incontact pads 32. The electrodes are defined as those portions ofelectrode traces 30 that are positioned within the sample-receivingchamber 24. Various configurations of the electrode system 26 may beused, as set forth hereafter. A suitable reagent system 33 overlies atleast a portion of the electrodes or electrode pairs 28 within thesample-receiving chamber.

The body cover 18 and the chamber cover 20 overlying the spacing layer14 define a slot 34 therebetween, the slot defining a vent openingcommunicating with the sample-receiving chamber to allow air to escapethe chamber as a sample fluid enters the chamber from the edge openingor fluid receiving opening 35. The test strip therefore includes adosing end 36 and a meter insertion end 38. The shape of the dosing endis typically distinguishable from the meter end so as to aid users. Inaddition, strip graphics are preferably used to further improve theintuitiveness of the strip design; e.g., arrow 31 indicates thedirection of insertion of the strip into the meter.

General Dimensions.

The test strip is a relatively small device that is dimensioned forcompactness and ease of storage and use. In a typical embodiment, thestrip length is on the order of 20 to 50 mm, preferably about 33 toabout 38 mm, in length, and 5 to 15 mm, preferably about 7 to about 9mm, in width. The distance from the slot or vent opening 34 to the edgeof the meter is sized to provide a “grab area” where there is no bloodpresent, and to guard against blood contamination of the meter contactarea, and therefore may be in the range of 5 to 35 preferably ≧13 mm.The length of the test strip portion (from the meter insertion end 38)that is inserted into the meter is preferably ≦6.0 mm along the longaxis of the test strip.

The preferred laminar construction of the test strip also provides adevice that is relatively thin. The minimal thickness of the stripallows ready packaging of the strip in appropriate containers that areconvenient for the user. For example, the overall thickness of the teststrip may be about 500 to 525 μm. The thickness of the test stripportion that is inserted into the meter contact may be about 250 μm.

Substrate

The test strip includes a base substrate 12 which comprises aninsulating material supporting the electrode system and othercomponents. Typically, plastics such as vinyl polymers, polyimides,polyesters, and styrenes provide the electrical and structuralproperties which are required. Further, because the test strip ispreferably mass producible from rolls of material, it is desirable thatthe material properties be appropriate to have sufficient flexibilityfor roll processing, while also giving a useful stiffness to thefinished strip. The base substrate can be selected as a flexiblepolymeric material such as polyester, especially high temperaturepolyester materials; polyethylene naphthalate (PEN); and polyimide, ormixtures of two or more of these. Polyimides are available commercially,for example under the trade name Kapton®, from E.I. duPont de Nemoursand Company of Wilmington, Del. (duPont). A particularly preferred basesubstrate material is MELINEX® 329 available from duPont.

Electrodes

Type.

The invention relates to an “electrochemical sensor”, which is a deviceconfigured to detect the presence of, and/or measure the concentrationof, an analyte by way of electrochemical oxidation and reductionreactions within the sensor. These reactions are transduced to anelectrical signal that can be correlated to an amount or concentrationof the analyte. The test strip therefore includes an electrode system 26comprising a set of measuring electrodes, e.g., at least a workingelectrode and a counter electrode, within the sample-receiving chamber.The sample-receiving chamber is configured such that sample fluidentering the chamber is placed in electrolytic contact with both theworking electrode and the counter electrode. This allows electricalcurrent to flow between the measuring electrodes to effect theelectrooxidation or electroreduction of the analyte.

In the context of the present invention, a “working electrode” is anelectrode at which analyte is electrooxidized or electroreduced with orwithout the agency of a redox mediator. The term “counter electrode”refers herein to an electrode that is paired with the working electrodeand through which passes an electrochemical current equal in magnitudeand opposite in sign to the current passed through the workingelectrode. The term “counter electrode” is meant to include counterelectrodes which also function as reference electrodes (i.e.,counter/reference electrodes).

Electrode Material.

The working and counter electrodes, and the remaining portions of theelectrode system, may be formed from a variety of materials, as known inthe art. The electrodes should have a relatively low electricalresistance and should be electrochemically inert over the operatingrange of the test strip. Suitable conductors for the working electrodeinclude gold, palladium, platinum, carbon, titanium, ruthenium dioxide,and indium tin oxide, and iridium, as well as others. The counterelectrode may be made of the same or different materials, e.g.,silver/silver chloride. In a preferred embodiment, the working andcounter electrodes are both gold electrodes.

Electrode Application.

The electrodes may be applied to the base substrate in any fashion thatyields electrodes of adequate conductivity and integrity. Exemplaryprocesses are well known in the art, and include, for example,sputtering, printing, etc. In a preferred embodiment, gold electrodesare provided by coating the base substrate and then removing selectedportions of the coating to yield the electrode system. A preferredremoval method is laser ablation, and more preferably broad field laserablation, as disclosed in U.S. patent application Ser. No. 10/601,144,filed on Jun. 20, 2003, entitled Method of Making a Biosensor, thedisclosure of which is hereby incorporated by reference.

Laser ablative techniques typically include ablating a single metalliclayer or a multi-layer composition that includes an insulating materialand a conductive material, e.g., a metallic-laminate of a metal layercoated on or laminated to an insulating material (discussed below). Themetallic layer may contain pure metals, alloys, or other materials,which are metallic conductors. Examples of metals or metallic-likeconductors include: aluminum, carbon (such as graphite), cobalt, copper,gallium, gold, indium, iridium, iron, lead, magnesium, mercury (as anamalgam), nickel, niobium, osmium, palladium, platinum, rhenium,rhodium, selenium, silicon (such as highly doped polycrystallinesilicon), silver, tantalum, tin, titanium, tungsten, uranium, vanadium,zinc, zirconium, mixtures thereof, and alloys or solid solutions ofthese materials. Preferably, the materials are selected to beessentially unreactive to biological systems; such materials include:gold, platinum, palladium, iridium, silver, or alloys of these metals orindium tin oxide. The metallic layer may be any desired thickness. In apreferred embodiment, the thickness is about 500 nm.

Configuration.

The electrode system may have a variety of configurations suited to theoperation of the test strip and meter. For any embodiment, the workingand counter electrodes preferably are positioned and dimensioned tominimize the volume of sample fluid required to cover them. It is alsopreferable that the electrodes be configured to maintain a current fluxof sufficient magnitude as to be measurable using a relativelyinexpensive hand-held meter.

By way of further example, a preferred embodiment includes a counterelectrode which extends around both sides of the working electrode. Thecounter electrode therefore has two elements, one in front of theworking electrode and the other behind the working electrode, as thesample fluid enters the sample-receiving chamber. More specifically, thecounter electrode includes elements 40 and 42 which extend across thesample-receiving chamber. Each of these elements is about 250 μm wide.The working electrode element 44 has a width of about 250 μm, and isspaced from each of the two counter electrode elements by about 255 μm.It will be appreciated that this is only one of a number ofconfigurations for the measuring electrodes.

The traces 30 and the contact pads 32 may be provided in a variety offashions consistent with their intended function relative to the teststrip. These components of the electrode system are preferably composedof the same material as the electrodes, and are preferably applied tothe base substrate in the same manner and simultaneously with theapplication of the electrodes. In a preferred embodiment, the traces andcontact pads are gold, and are formed by laser ablation, particularly asdescribed in U.S. patent application Ser. No. 10/601,144, filed on Jun.20, 2003, entitled Method of Making a Biosensor, the disclosure of whichis hereby incorporated by reference. However, alternate materials andmethods of application may be employed.

Chemistry

Reagent Composition.

The test strip includes a chemical reagent within the sample-receivingchamber for reacting with the test analyte to produce theelectrochemical signal that represents the presence of the analyte inthe sample fluid. The reagent layer can include a variety of activecomponents selected to determine the presence and/or concentration ofvarious analytes. The test chemistry is therefore selected in respect tothe analyte to be assessed. As is well known in the art, there arenumerous chemistries available for use with each of various analytes.For example, in one preferred embodiment, the test strip of the presentinvention can include one or more enzymes, co-enzymes, and co-factors,which can be selected to determine the presence of glucose in blood. Theselection of an appropriate chemistry is therefore well within the skillin the art, and further description herein is not required in order toenable one to make and use the test strips with various analytes.

Adjuvants.

In conventional fashion, the reagent chemistry may include a variety ofadjuvants to enhance the reagent properties or characteristics. Forexample, the chemistry may include materials to facilitate the placementof the reagent composition onto the test strip and to improve itsadherence to the strip, or for increasing the rate of hydration of thereagent composition by the sample fluid. Additionally, the reagent layercan include components selected to enhance the physical properties ofthe resulting dried reagent layer, and the uptake of a liquid testsample for analysis. Examples of adjuvant materials to be used with thereagent composition include thickeners, viscosity modulators, filmformers, stabilizers, buffers, detergents, gelling agents, fillers, filmopeners, coloring agents, and agents endowing thixotropy.

In a preferred embodiment of the test sample, the majority of thechamber is hollow before use. In the very small sample chamber of thetest strips according to the present invention, it is preferable thatthe reagent layer be thin and uniform. Since the sample-receivingchamber is very small, less than about 1 μl, the depth or verticalheight of the chamber is small. Consequently, the reagent layer shouldnot occupy the majority of the internal cavity of the chamber. Thereagent layer should be sufficiently thin to leave ample space for thetest sample in the chamber. Further, the liquid test sample will hydrateor dissolve the thin reagent layer more quickly. As discussed in theabove reaction scheme, the mediator and mediator redox products diffusethrough and within the reagent layer/gradient to the electrodes. Thereactive components and intermediates will have a short distance todiffuse through a thin reagent, therefore, diffusion to the electrodeswill occur in less time. Additionally, the capture efficiency ofmediator redox products at an electrode will be greater for a thin layerof enzyme than a thick layer.

Conversely, a thick reagent layer will take more time for the liquidtest sample to hydrate or dissolve, and a thick reagent layer willincrease the time that it takes for the mediator/mediator redox productsto approach the electrodes. This can delay the time to determine theanalyte concentration and introduce errors into the determination.

It is preferred that the reagent layer have a uniform thickness.Thickness inhomogeneity can lead to variability in determining theanalyte concentration. In a preferred embodiment, the reagent layer hasa uniform thickness throughout the entire sample receiving chamber. Inthis preferred embodiment, the reagent layer is not thicker around theperimeter of the sample receiving chamber adjacent the vertical sidewalls that define the chamber than in the central portion of thechamber. Consequently, the reagent layer does not exhibit a meniscusprofile.

The reagent composition is formulated as a viscous solution that can bedeposited in a thin, uniform layer on the base layer. The reagentcomposition includes thickeners and thixotropic agents to enhance thephysical properties of the reagent layer. The thickeners are selected toprovide a thick, liquid matrix having the remaining componentshomogeneously dispersed therein. The thickening and thixotropic agentsalso inhibit the liquid or semi-paste material from running or spreadingover the surface of the base layer after it has been deposited andbefore it dries. After the reagent composition is deposited, it quicklydries to a readily hydratable matrix.

The reagent composition is provided to dry rapidly either with airdrying or heat drying. After drying, the deposited reagent layerexhibits a thickness of between about 1 micron and about 20 microns.More preferably, the dried reagent layer exhibits a thickness of betweenabout 2 microns and about 6 microns.

The reagent composition can be deposited on the test strip surface usinga variety of coating methods including curtain coating, hot meltcoating, rotary screen coating, doctor blade or air knife coating, Meyerbar coating, and reverse roll coating techniques. These techniques areknown to those skilled in the art. Preferably, the reagent layer isdeposited on the flexible web as a wet composition at a thickness ofbetween about 40 μm and about 100 μm. More preferably, the reagentcomposition is deposited as a wet composition at a thickness of betweenabout 60 μm and about 80 μm. The composition may be applied as auniformly thin layer of a reagent directly on top of the measuringelectrodes and along the length of a web of multiple test strips, as acontinuous narrow band. In preferred embodiments, the narrow band has awidth of between about 7 mm and 8 mm and a dry thickness of betweenabout 3 um and about 20 um. The composition may also be applied ontoother electrodes that may reside in the sample-receiving chamber,depending on the desired functionality of such extraneous electrodes.

Spacing Layer

Configuration.

The test strip includes a spacing layer 14 which overlies the basesubstrate and which in part defines the sample-receiving chamber. Inparticular, the spacing layer 14 includes a void portion 22substantially defining the height and the perimeter of thesample-receiving chamber 24. The void portion 22 is conveniently placedto have an edge opening whereby the sample fluid is contacted with theedge opening to enter into the sample-receiving chamber. The edgeopening preferably is located at the end of the test strip, although itwill be appreciated that placement on a side edge is also useful.

Materials.

The spacing layer 14 may be made of any material useful for fabricationwith the test strip. Because the spacing layer partially defines theheight of the sample-receiving chamber, the material should havesufficient strength at thicknesses appropriate to the desired height ofthe chamber. Another function of the spacing layer is to protect theelectrode traces that extend along the upper surface of base substrate12. The material should also be readily attached to the base substrateand the cover materials, either by heat-sensitive or pressure-sensitiveadhesives, or other means such as heat or laser welding. Examples ofsuitable materials include a 100 μm PET, PEN foil coated or combinedwith adhesives such as ARCare 90132 from Adhesives Research Inc.

Covering Layer

Configuration.

A covering layer 16 is received over and attached to the spacing layer14. One function of the covering layer is to form the top surface of thesample-receiving chamber. Another function is the provision of ahydrophilic surface to aid in acquisition of the test sample. Inaddition, the covering layer 16 preferably defines a vent opening 34that allows air to escape from the interior of the chamber as the samplefluid enters and moves into the sample-receiving chamber.

The covering layer can be formed as a unitary piece with slot 34′ formedas a recess on the underside thereof, as shown in FIG. 1B. For massproduction purposes, slot 34′ would be substantially straight as shownand extend across the entire width of the test strip, such that airwould vent from the sample receiving chamber 24 to the vent openingsformed on opposite lateral sides of the test strip. However, the slotcould comprise a groove or recess that only extends from the chamber 24to one side of the test strip, although such configuration is notpreferred for mass production purposes.

Another alternate embodiment is shown in FIG. 1C, in which chamber cover20 “overlaps” body cover 18. In this arrangement, a small end portion 37of cover layer 20 is bent upwardly and extends across the edge of bodycover 18. A slot 34″ is thereby formed having roughly a triangularshaped cross section as can be seen at the edges of the strip, at whichthere are triangular shaped openings that allow air to escape. In this“overlap” arrangement, the precise placement of the chamber cover 20with respect to body cover 18 along the lengthwise direction of thestrip is not critically important. That is, the amount of chambercovering material overlapping body cover 18 can vary without affectingthe dimensions or placement of the slot. This has advantages inmanufacturing, as will become apparent with reference to the discussionbelow.

Preferably, body cover 18 and chamber cover 20 comprise two separatemembers for ease in fabrication and in forming the vent opening. Bodycover 18 and chamber cover 20 are both disposed in substantially thesame horizontal plane. The chamber cover 20 substantially covers thevoid portion 22 of the spacing layer, and forms the top of thesample-receiving chamber. The chamber cover preferably includes ahydrophilic coating or treatment 21 on its underside, as described inmore detail below. The body cover and the chamber cover are positionedend to end in the lengthwise direction along the test strip and includeslot 34 therebetween as shown in FIG. 1A. The slot is located adjacentthe interior end of the void portion 22 of the spacing layer, and in thepreferred embodiment in FIG. 1A, forms a small gap that spaces chambercover 20 from body cover 18. The gap constitutes the vent opening 34 incommunication with the sample-receiving chamber. Slot 34 issubstantially straight and extends across the width of test strip 10.Slot 34 is oriented substantially perpendicular to the longitudinal orlengthwise axis of test strip 10. Sample fluid entering thesample-receiving chamber will expel air through the vent opening definedby slot 34. If the slot be formed as a gap, some or most of the airexpelled will exit from the top of the test strip.

The slot is located at a position relative to the sample-receivingchamber that is interior of the location of the electrode system 26.Sample fluid entering the sample-receiving chamber will progress as faras the vent opening, but no further. When viewed from the top, the slotprovides a visual indication of a “fill-line,” as described herein. Theplacement of the vent opening therefore assures that sufficient samplefluid can be received to fully cover the electrode system. At the sametime, the placement of the vent opening will inhibit continued wickingof the sample fluid beyond the region of the electrode system.

The formation of the slot and vent opening by the spacing of the bodycover and the chamber cover is further advantageous because it avoidsthe need to otherwise form an aperture in the covering layer or baselayer. In the prior art, it has been an approach to form the ventopening by punching a hole in either the top or bottom film forming thesample-receiving chamber, which presents fabrication issues because ofthe need to precisely locate the hole relative to the sample-receivingchamber. While this approach is also suitable for a test strip, thepreferred design described herein avoids the need to align the ventopening laterally relative to the test strip. Further, the presentdesign is well suited to mass production of the test strips by rollprocessing techniques, as described hereafter.

At the same time, the vent construction may be made in a manner toinhibit the wicking of sample fluid laterally along the slot beyond themiddle area that overlies the sample receiving chamber 24. For example,the body cover is preferably secured to the spacing layer by means of anadhesive 46, as shown in FIG. 3. The use of a hydrophobic adhesive willinhibit blood, interstitial fluid, and other aqueous liquids from movingalong the laterally-extending slot by capillary action. The entire bodycover, or portions adjacent to the vent opening, may also be hydrophobicto inhibit wicking. Materials and methods for providing hydrophobicproperties for a surface of a material are well known in the art. Thechamber cover may be secured to the spacing layer by the same ordifferent adhesive than adhesive 46, as explained below.

Adhesive 49 secures the spacing layer to the base substrate 12. Adhesive46, as well as adhesive 49 and the material for spacing layer 14, areall formed of hydrophobic material in the illustrated embodiment. Assuch the vertical walls of the capillary chamber formed in strip 10 arehydrophobic. By contrast, the floor of the chamber is covered with ahydrophilic reagent and the underside of layer 20 is coated with ahydrophilic coating 21 (FIG. 2). In other words, the horizontal surfacesin the capillary are hydrophilic while the vertical surfaces arehydrophobic. This has been found to promote good wicking of the sampleinto the capillary chamber, yet prevents unwanted migration of thesample laterally from the chamber, e.g., between the spacer layer andthe base substrate.

Materials.

The body cover and chamber cover may be made of any materials useful forfabrication with the test strip. The materials for the body cover andthe chamber cover may be the same or different. The materials should bereadily attached to the spacing layer, either by heat-sensitive orpressure-sensitive adhesives, or other means such as heat or laserwelding. Examples of suitable materials for both the chamber cover andbody cover include approximately 127 μm thick foil of PET. The chambercover preferably includes a hydrophilic layer 21 as disclosed in WO02/085185, ARFlow® 90191 from Adhesives Research Inc.

The covering layer 16 may also be used to facilitate viewing of thesample fluid as it enters the sample-receiving chamber. This isaccomplished by providing a contrast in color or shading between thechamber and the surrounding area. For example, in one approach theportion of the spacing layer 14 that surrounds void 22 is provided witha color that contrasts with the color of the bottom of thesample-receiving chamber, e.g., the color of the chemical reagent layerpositioned at the bottom of the chamber. This contrasting color may beprovided, for example, by the application of an ink or other coloringagent to the portions of the spacing layer adjacent the sample-receivingchamber. A colored section 23 of layer 14 is pictured in FIG. 2. Thechamber cover 20 is then provided as a transparent or translucentmaterial that allows the user to view the chamber and the adjacentspacing layer. As sample fluid enters from the edge of the test strip,the user is able to observe its progress as it moves by capillary actiontoward the vent opening. This type of feature is further described inU.S. Pat. No. 5,997,817, issued to Crismore et al. on Dec. 7, 1999, andis hereby incorporated by reference.

Capillary

The sample-receiving chamber formed by the base substrate, spacing layerand chamber cover essentially comprises several sections into which thesample fluid will travel. A first, entry section 48 extends from theedge opening to the area of the measuring electrode system. A second,test section 50 extends through the area of the electrode system. Athird section 52 extends from the measuring electrode system to the ventopening. It will be appreciated that the testing of the sample fluidoccurs in the area of the electrode system in the test section. However,the sample fluid will also fill the other sections of the chamber in thecourse of filling the test strip.

Dimensions.

The height and width of the sample-receiving chamber are selected basedupon a variety of considerations, including the fluid being tested andthe analyte at issue. For example, the chamber dimensions are preferablysized to promote capillary flow of the test fluid into the chamber.Preferred chamber heights for use with blood, for example, are fromabout 50 μm to about 200 μm, and most preferably from 120 to 180 μm. Ina preferred embodiment, the chamber height is about 150 μm. The width ofthe chamber may similarly be selected to match a desired sample fluidand analyte. For example, the chamber should be sufficiently wide toexpose a desired amount of the working and counter electrodes, andshould be narrow enough to avoid the requirement of an undue amount ofsample fluid for testing. The width of the sample-receiving chamber andthe width of the working electrode define the area of the workingelectrode. The area represents a further design consideration as itrelates to signal amplitude and instrumentation design.

Volume.

The sample-receiving chamber is preferably provided as having a minimalvolume, in order to reduce the amount of sample fluid needed forconducting a test. The overall sample-receiving chamber, including allof the three sections extending from the edge opening to the ventopening, has a total volume that can be considered to be a factor of thearea of the chamber from the edge to the vent, and the height of thechamber from the base substrate to the chamber cover 20. However, the“net chamber volume” comprises the volume of sample fluid required tofill this space. The net chamber volume of the sample-receiving chamberwill be the equivalent of the total chamber volume less the volumeoccupied by the electrodes, the reagent, and perhaps other items such asa sorbent material, if included.

As previously indicated, the volume of the overall sample-receivingchamber is comprised of the volumes attributable to the three sectionsof the chamber. Each of the sections is generally sized to be as smallas practical for the operation of the test strip. However, there areconsiderations, and possibly other functions, that will impact on thesize of each section.

The chamber volumes are a factor of both height and area. The height isa result of the thickness of the spacing layer and the thickness of theadhesives used to secure the spacing layer to the other layers. Forexample, the base substrate and the chamber cover are attached toopposite sides of the spacing layer. One method of attachment is theheat or laser sealing of the materials. It is preferred, however, toattach these layers by the use of suitable adhesives, such asheat-sensitive or pressure-sensitive adhesives. In this approach, theheight of the sample-receiving chamber, i.e., the distance between thefacing surfaces of the bottom substrate and the chamber cover, will beimpacted by the thickness of the adhesive layers. As shown in FIG. 3,chamber 24 is bounded on its bottom side by reagent layer 33 and its topside by chamber cover 20. However, adhesive layers 46 and 49 as well asspacing layer 14 define the total height of chamber 24.

Further, in a preferred embodiment, the reagent layer 33 extends betweenbase substrate 12 and spacing layer 14 and indeed extends the entirewidth of the test strip, as described below. The height of the chambermay therefore also be increased due to the presence of the reagent layerunderlying the spacing layer. In this embodiment, and if adhesive isemployed, it has been found that the adhesive may combine with the testreagent, at least to an extent that causes the adhesive to fill somewhatinto and around the reagent. The heights of the reagent and adhesivelayers therefore are not necessarily additive in the final test strip.Rather, the height of the resulting space between the base substrate andthe spacing layer is somewhat less than the combination of the heightsof the separate reagent and adhesive layers prior to lamination.

It has also been found that the combination of the adhesive and thereagent advantageously helps to create a seal along the edge of thesample-receiving chamber. This inhibits sample fluid from wicking intothe reagent material present in the space between the base substrate andthe spacing layer in the time frame necessary for performing a test.

The first entry section is available to receive the sample fluid anddirect it to the measuring electrodes. This section can be quite smallin size, and may comprise only a short segment of the chamber. Thelength of this section is preferably less than 1200 μm.

The second testing section includes the test or measuring electrodes,and is also sized to require a minimal volume of sample fluid. A primaryfactor controlling the size of this second section will be the type,number, size, signal strength, and configuration of the measuringelectrodes. The length of this section is preferably about 1260 μm. Apreferred volume is about 0.265 μL, based on a capillary height of 0.15mm, and a capillary width of 1.4 mm.

The sample fluid moves past the measuring electrodes and into the thirdsection. This provides assurance, and preferably allows for specificconfirmation, that the measuring electrodes are properly wetted. Thisconfirmation may be by visual observation by the user, or by automaticdetecting means. For example, dose sufficiency electrodes may be placedin this section to detect when the sample fluid has progressed into thissection to a point that the wetting of the measuring electrodes isassured. This can be used as a trigger for initiating the application ofthe potential to the electrodes. The length of this section ispreferably 50 to 500 μm, and more preferably 255 to 400 μm. The volumeis preferably 0.01 to 0.1 μL, and more preferably 0.05 to 0.08 μL.

In a preferred embodiment, the overall net chamber volume of thesample-receiving chamber is less than about 1 μL, and is more preferablyless than about 0.5 μl. Desirable ranges for the net chamber volume ofthe sample-receiving chamber include volumes from about 0.15 to about1.4 μL, more preferably from about 0.4 to about 0.7 μl.

Sorbent.

The sample chamber may be otherwise empty, which is preferred, or mayalternatively include a sorbent material. Suitable sorbent materialsinclude polyester, nylon, cellulose, and cellulose derivatives such asnitrocellulose. A sorbent material could be included to facilitate theuptake of the sample fluid by assisting in wicking the fluid into thechamber. The use of a sorbent material would also serve to furtherreduce the void volume of the sample-receiving chamber for reception ofthe sample fluid.

Fill Method.

The preferred method of filling the sample chamber is by capillaryaction. In addition, the filling of the test strip can be augmented byother means, such as applying a pressure on the sample fluid to push itinto the sample chamber, and/or creating a vacuum on the sample chamberto pull the sample fluid into the chamber.

Hydrophilic Coating.

For purposes of capillary filling of the sample-receiving chamber,various approaches are available to facilitate the movement of thesample fluid into the chamber. For example, any or all of the surfacesdefining the chamber may be selected or treated to improvehydrophilicity. Such treatment may comprise the use of known hydrophilicmaterials, application of a hydrophilic material onto the surface, ortreatment of the surfaces to increase hydrophilicity, as describedbelow. In addition, the reagent composition may be formulated to bereadily hydrated and to encourage filling of the sample-receivingchamber. As previously indicated, a sorbent may also be used.

Testing for Analyte

The electrochemical sensor is operated by applying a suitable potentialor series of potentials across the working and counter electrodes, andacross the dose sufficiency electrodes. When a mediator is used, themagnitude of the required potential across the working and counterelectrodes will be dependent on the redox mediator. Moreover, thepotential at the electrode where the analyte is electrolyzed istypically large enough to drive the electrochemical reaction to or nearcompletion, but the magnitude of the potential is preferably not largeenough to induce significant electrochemical reaction of interferants.For glucose, for example, an applied potential difference typically isbetween about +100 mV and about +550 mV when using a DC potential. Whenusing AC potentials these can be typically be 5 to 100 mV RMS.

A potential may be applied before or after the sample begins to enterthe sample-receiving chamber. However, a potential is preferably appliedafter the sample has entered the chamber, and more preferably after ithas been determined that there is a sufficient amount of sample in thesample-receiving chamber for conducting a test. The timing of theapplication of a potential may be triggered in a variety of fashions,including visual observation by the user, a time delay followingsampling of the fluid to the test strip, or upon electrical or otherautomated detection of a sufficient amount of sample fluid in thechamber. The visual and electrical alternatives also may act asredundant failsafes to assure proper operation of the device.Preferably, the test strip and system utilize separate detecting means,such as dose sufficiency electrodes, for determining when the fluidsample has sufficiently filled the chamber.

When a potential is applied and the sample fluid is in thesample-receiving chamber, an electrical current will flow between theworking electrode and the counter electrode. The current can be a resultof the electrolysis of the analyte in the sample fluid when a potentialof sufficient magnitude is applied. In this case electrochemicalreaction occurs via the redox mediator, generally as previouslydescribed. In the case where small amplitude potential is applied,particularly in the case of AC potentials, the current is produced notnecessarily by electrolysis, but by ionic motion and response of thedielectric in the sample chamber. Those skilled in the art willrecognize that there are many different reaction mechanisms that willachieve the same result.

Control Solution

A test may be applied to the test strip after dosing to confirm that acontrol solution, and even that the correct control solution, has beenadministered. The control solutions aid the user in confirming that theentire system is functioning within design specifications, and that thetest strips have not been stored improperly or otherwise mistreated.Acceptable strips will recover values within specified tolerance rangesfor the particular strip lot being tested. The tolerance ranges inquestion will be published for each strip lot on the container label.

Method of Making Strip

In a preferred embodiment, the sensor comprises a multi-layered,laminate test strip 10. As previously described, the laminate includes abase substrate 12, a spacing layer 14, and a covering layer 16. Thesecomponents may be assembled in various ways. For example, the componentsmay be assembled by use of adhesives, heat sealing, laser welding, and avariety of other suitable techniques appropriate for securing theadjacent materials. The test strips are preferably assembled in a largenumber on a single sheet or web, and the strips are thereafter separatedfor storage and use.

The laminate test strip may be assembled sequentially by successivelylaying down one layer at a time. Alternatively, the test strip can beprepared by assembling and processing individual components or layers,which are then laminated together to provide the functional test strip.In one preferred form, two or more basic components of the test stripare prepared simultaneously. Then in one or a series of assembly orlaminating steps, the basic components are combined to produce the teststrip, which may or may not require further processing. In a preferredembodiment, the test strip is assembled from three basic components: ametallized substrate preferably with a reagent layer coated on metallicelectrodes defined on the substrate, a spacing layer having a cavitypreformed therein, and one or more top or cover layers.

With such small dimensions for the sample-receiving chamber, thecharacteristics of the reagent layer can have a significant impact onthe operation of the test strip, particularly in view of hydration andmixing characteristics. The reproducibility of the quantity, location,thickness and other properties of the reagent layer is thereforeimportant. It is therefore desirable for the composition to includematerials which specifically enhance the physical characteristics, suchas the uniformity and flatness, of the applied layer.

In one particular aspect, the test strip includes a unique manner ofincorporating the reagent. The reagent is placed in the sample-receivingchamber at least on the working electrode, and preferably also on thecounter electrode. The reagent may be applied to the test strip in avariety of fashions as is well understood in the art. In a preferredembodiment, the reagent composition is applied as a thin coating overthe electrodes supported on the base substrate.

More particularly, the reagent is placed onto the base substrate in amanner that positions the reagent composition between the base substrateand the spacing layer. This manner of application helps to make thereagent layer more flat and uniform in thickness. In contrast, aprocedure of the prior art has been to first prepare the reaction wellor cavity, and to then fill the reagent into the well. However, this canresult in a more uneven reagent layer due to phenomena such as theformation of a meniscus at the perimeter of the well. This in turn cancause the reagent to have a different thickness adjacent to the sidewalls of the reaction well than in the interior portion, which can causeinconsistency in the filling of the chamber, prolonged dissolutionintervals, and inconsistent mixing of the reagent with the sample fluid,and the ultimate test results. By placing the reagent onto the basesubstrate before the spacing layer is added, there is no meniscus effectto disrupt the even layering of the reagent as it dries on the basesubstrate. In addition, this method of application facilitates the massproduction of the test strips.

Referring to the drawings, the test strip 10 is shown as including areagent layer 33 that extends between the bottom substrate 12 and thespacing layer 14. More particularly, the reagent forms a layer 33 whichcovers both the top surface of the bottom substrate 12 and theelectrodes 28. The reagent covers at least the working electrode, andpreferably also the counter electrode. In the most preferred embodiment,the reagent layer extends the full width of the test strip. The reagentlayer also preferably extends from the end edge to the vent opening. Thereagent layer thus extends under the spacing layer and is sandwichedbetween the spacing layer and the base substrate.

The reagent composition is applied to the bottom or base substrate inany suitable fashion that provides a desired and uniform layer whichwill ultimately extend under the spacing layer. The reagent ispreferably applied in a continuous coating directly onto the bottomsubstrate, and onto the electrodes received thereon. As describedhereafter, the reagent composition is most preferably applied in thecourse of producing a large quantity of test strips on a webbing ofmaterial. In this manner, the reagent may be applied in the way of acontinuous stripe of material that extends over a substrate roll that islater separated into individual test strips. The reagent composition isallowed to dry or otherwise set up and the spacing layer is appliedthereover.

In a related aspect, a preferred manner of securing the spacing layer tothe bottom substrate is the use of an adhesive. In addition to securingthe layers together, it has been found that the adhesive willsufficiently engage with the reagent composition as to help to seal thespace between the bottom substrate and the spacing layer. The adhesivespreferably placed on the spacing layer, which is laminated onto the basesubstrate. The adhesive thereby contacts the portion of the reagentwhich extends under the spacing layer.

Although the spacing layer of the illustrated embodiment is formed fromMelinex® material with adhesives on both sides thereof, it is alsopossible to form spacing layer 14 as a continuous adhesive material,such as a double-sided tape.

In a further aspect, a preferred embodiment is described in which theanalyte is glucose. In the case of glucose, the active components of thereagent composition will typically include an oxidoreductase, such as anenzyme for glucose; optionally a co-enzyme or co-factor; and a redoxmediator. These components are typically dissolved or suspended in amatrix. The liquid test sample hydrates or dissolves the matrix, and theanalyte diffuses through the matrix to react with one or more of theactive components. Typically, the enzyme oxidizes the glucose in thetest sample to gluconolactone and/or gluconic acid. The mediator, inturn, reacts with or oxidizes the reduced enzyme, and consequently themediator is reduced in the process. The reduced mediator can be detectedat one of the electrodes on the test strip.

In a specific example of an oxidation/reduction reaction scheme usefulfor detecting glucose in human blood, a test sample containing glucosereacts with an enzyme such as Glucose-Di-Oxidoreductase (Gluc-Dor), andoptionally a co-enzyme or cofactor such as pyrrolo-quinoline-quinone(PQQ), in the presence of a redox mediator. The mediator may include,for example, benzoquinone, transition metal complexes, e.g., potassiumferricyanide, osmium derivatives (e.g., osmium bipyridyl complexes suchas described in WO 98/35225) and nitrosoanaline derivatives (see U.S.Pat. No. 5,286,362). This produces the oxidized form of the analyte,gluconolactone, and the reduced form of the redox mediator. The mediatorthereafter shuttles the redox equivalent of mediator product, thereduced mediator, to the electrode surface by diffusion. There themediator is oxidized quantitatively at a defined anodic potential, andthe resulting current is related to the apparent glucose concentration.

A representation of the reaction sequences for this reaction systemusing a nitrosoaniline derivative is provided below in Equation 1.

As shown, the nitrosoaniline derivative,o-methoxy-[N,N-bis-(2-hydroxyethyl)]-p-nitrosoaniline, initially existsas a mixture of two isomers, or tautomers, in equilibrium with eachother. Reaction of Gluc-Dor with glucose in the test sample yieldsgluconolactone and the reduced form of Gluc-Dor (Gluc-Dor.2H⁺). Thereduced form of Gluc-Dor (Gluc-Dor.2H⁺) reacts rapidly with thenitrosoaniline derivative, which is reduced and which regeneratesGluc-Dor. The reduced nitrosoaniline derivative then undergoeshydrolysis to form quinonediimine (QD). In a second enzymatic, redoxreaction, Gluc-Dor reacts with glucose to yield another molecule ofGluc-Dor.2H⁺ and gluconolactone. The Gluc-Dor.2H⁺ reacts with (isoxidized by) quinonediimine to regenerate Gluc-Dor, and produces aphenylene diamine derivative (PD). PD is then oxidized at the workingelectrode to produce a current related to glucose concentration.Additionally, at the counter electrode QD can be reduced to PD.

Adjuvants.

With such small dimensions for the sample-receiving chamber, thecharacteristics of the reagent layer can have a significant impact onthe operation of the test strip, particularly in view of hydration andmixing characteristics. The control and reproducibility of the quantity,location, width, thickness, and other properties of the reagent layerbecome more important as the chamber volume decreases and test timediminishes. It is therefore desirable for the composition to includematerials that specifically enhance the physical characteristics, suchas the uniformity and flatness, of the applied layer. Additionally, themethod of application can impact the physical characteristics, control,and reproducibility of the reagent layer.

The reagent composition can therefore also include a variety ofadjuvants to enhance the reagent properties or characteristics. Forexample, the composition may include adjunct materials to facilitate theplacement of the reagent composition onto the test strip and to improveits adherence to the strip. The composition can also include materialsto increase its rate of hydration and/or its increase its influence onthe capillary action to fill the chamber with the test sample. Examplesof addjunct materials to be used with the reagent composition includethickeners, viscosity modulators, film formers, stabilizers, buffers,detergents, gelling agents, fillers, film opening agents, coloringagents, and agents endowing thixotropy.

The adjuvant materials or components can impact the application,reproducibility and physical properties of the reagent layer. Theadjunct materials can include one or more of the following:

Thickeners may include, for example, (1) starches, gums (e.g., pectin,guar gum, locust bean (carob seed) gum, konjac gum, xanthan gum,alginates, and agar), casein, gelatin, and phycocolloids; (2) celluloseand semi-synthetic cellulose derivatives (carboxymethyl-cellulose,methyl cellulose, hydroxymethylcellulose, hydroxyethylcellulose,methylhydroxyethylcellulose); (3) polyvinyl alcohol andcarboxy-vinylates; and (4) bentonite, silicates, and colloidal silica.Preferred thickeners include a combination of a xanthan gum sold underthe trade name Keltrol F by CP Kelco US, Inc., and carboxylmethylcellulose sold under the trade name AQUALON® CMC 7F PH by Hercules Inc.,Aqualon Division.

Film forming and thixotropic agents useful in the reagent compositioninclude polymers and silica. Preferred thixotropic agents include silicasold under the trade name Kieselsaure Sipemate FK 320 DS by Degussa AG.Preferred film forming agents include polyvinylpyrrolidone, sold underthe trademark polyvinylpyrrolidon Kollidon 25, by BASF, and polyvinylpropionate dispersion.

Stabilizers for the enzyme in the reagent can be selected fromsacchhrides and mono-or di-fatty acid salts. Preferred stabilizersinclude trehalose sold under the trade name D-(+)-Trehalose dihydrate bySigma Chemical Co. and sodium succinate.

Detergents can be selected from water-soluble soaps, as well aswater-soluble synthetic surface-active compounds such as alkali, earthalkali or optionally substituted ammonium salts of higher fatty acids,e.g., oleic or stearic acid, mixtures of natural fatty acids, forexample, from coconut or tallow oil, fatty sulphates, esters ofsulphonic acids, salts of alkyl sulphonic acids taurine salts of fattyacids, fatty acid amides, and ester amides. Preferred detergents for thepresent invention include an ester amide, n-octanoyl-N-methylglucamide,sold under the trade name Mega-8 by Dojindo Molecular Technologies,Inc., and a fatty acid salt, N-methyl oleyl taurate sodium salt, soldunder the trade name Geropon T77 by Rhodia HPCII (Home, Personal Careand Industrial Ingredients).

It should be understood that one or more of the specific additives abovedescribed can exhibit additional properties and consequently could becategorized in one or more of the classes above noted. Mediator.

A mediator for use in the reagent composition can be selected as anychemical species (generally electroactive) which can participate in areaction scheme involving an enzyme, an analyte, and optionally acofactor, and reaction products thereof, to produce a detectableelectroactive reaction product. Typically, participation of the mediatorin the reaction involves a change in its oxidation state (e.g., areduction), upon interaction with any one of the analyte, the enzyme, ora cofactor, or a species that is a reaction product of one of these(e.g., a cofactor reacted to a different oxidation state). A variety ofmediators exhibit suitable electrochemical behavior. A mediator canpreferably also be stable in its oxidized form, may optionally exhibitreversible redox electrochemistry, can preferably exhibit goodsolubility in aqueous solutions, and preferably reacts rapidly toproduce an electroactive reaction product. Examples of suitablemediators include benzoquinone, meldola blue, other transition metalcomplexes, potassium ferricyanide, osmium derivatives (see WO 98/35225)and nitrosoanaline-based mediators (see U.S. Pat. No. 5,286,362). In apreferred embodiment, the reagent composition utilizes anitrosoaniline-based chemistry.

Preferred mediators includeN-(2-hydroxyethyl)-N′-p-nitrosophenyl-piperazine,N,N-bis-(2-hydroxyethyl)-p-nitrosoaniline,o-methoxy-[N,N-bis-(2-hydroxyethyl)]-p-nitrosoaniline,p-hydroxynitrosobenzene, N-methyl-N′-(4-nitrosophenyl)-piperazine,p-quinone dioxime, N,N-dimethyl-p-nitrosoaniline,N,N-diethyl-p-nitrosoaniline, N-(4-nitrosophenyl)-morpholine,N-benzyl-N-(5′-carboxypentyl)-p-nitrosoaniline,N,N-dimethyl-4-nitroso-1-naphthylamine,N,N,3-trimethyl-4-nitrosoaniline, N-(2-hydroxyethyl)-5-nitrosoindoline,N,N-bis-(2-hydroxyethyl)-3-chloro-4-nitrosoaniline,2,4-dimethoxy-nitrosobenzene, N,N-bis-(2-methoxyethyl)-4-nitrosoaniline,3-methoxy-4-nitrosophenol,N-(2-hydroxyethyl)-6-nitroso-1,2,3,4-tetrahydroquinoline,N,N-dimethyl-3-chloro-4-nitrosoaniline,N,N-bis-(2-hydroxyethyl)-3-fluoro-4-nitrosoaniline,N,N-bis-(2-hydroxyethyl)-3-methylthio-4-nitrosoaniline,N-(2-hydroxyethyl)-N-(2-(2-methoxyethoxy)-ethyl)-4-nitrosoaniline,N-(2-hydroxyethyl)-N-(3-methoxy-2-hydroxy-1-propyl)-4-nitrosoaniline,N-(2-hydroxyethyl)-N-(3-(2-hydroxyethoxy)-2-hydroxy-1-propyl)-4-nitrosoaniline,N-(2-hydroxyethyl)-N-(2-(2-hydroxyethoxy)-ethyl)-4-nitrosoaniline.Particularly preferred mediators according to the present inventioninclude N,N-bis-(2-hydroxyethyl)-p-nitrosoaniline,o-methoxy-[N,N-bis-(2-hydroxyethyl)]-p-nitrosoaniline, andN-(2-hydroxyethyl)-N-(2-(2-hydroxyethoxy)-ethyl)-4-nitrosoaniline.

An exemplary reagent composition is listed below in Table I. TABLE IAmount % solids Components Function Abs. w/w. Note. Keltrol F Thickener11.60 g 0.24% Carboxy methylcellulose Thickener 27.24 g 0.57%Kieselsäure Sipernat Film Opener 97.01 g 2.01% 320 DSPolyvinylpyrrolidine Film Former 89.33 g 1.85% PVP K25 Propiofan FilmFormer 257.09 5.34% GlucDOR Apo-Enzyme 19.127 g  0.40% 0.673 MU/gpyrrolo-quinoline quinine Co-Factor 0.5329 g  0.01% (PQQ) Na-SuccinateStabilizer 23.23 g 0.48% Trehalose Stabilizer  23.6 g 40.49%  KH₂PO₄Buffer 12.02 g 0.39% K₂HPO₄ × 3 H₂O Buffer 43.43 g 0.90% NitrosoanilineMediator 41.26 g 0.86% Mega 8 Detergent 13.23 g 0.27% Geropon T77Detergent 1.405 g 0.03% KOH 5N Adjust 36.47 g 0.76% Buffer Water total4114.52 g  Sum 4817.80 g  Solids 14.6%Mixing.

The components of the reagent composition are admixed with water toprovide a homogeneous, viscous suspension. The order of addition is notcritical for the invention. A sufficient amount of the buffer solutionis added to maintain the reagent composition at a pH of about 7.Typically the selected components are pre-mixed with water to provide avariety of stock solutions that can be combined to yield the finalreagent composition. For example, a buffer solution can be prepared bycombining the phosphate salts and, optionally, the sodium succinate.Other stock solutions include: the thickening agents, i.e., Keltrol Fand the carboxymethyl cellulose; the surfactants, i.e., Geropon T77 andMega 8; the enzyme and co-enzyme or cofactor; and the mediator.

The following provides an example of the preparation of a reagentcomposition. The reagent composition can be prepared by first preparingthe following stock solutions: Buffer Solution pH 6.9 to 7.1 Amount (gm)H₂O 1214.62 KH₂PO₄ 18.27 K₂HPO₄ 43.43 Na succinate 23.23

Keltrol F Solution Amount (gm) H₂O 287.06 Buffer Solution 101.35 KeltrolF 11.60

Carboxymethylcellulose (CMC) Solution Amount (gm) H₂O 1334.76 Na CMC¹27.241. Na CMC is a sodium salt of carboxymethyl cellulose sold by HerculesInc.,

Aqualon Division Silica Suspension Amount(gm) H₂O 722.99 Sipernat 320¹¹Kieselsäure Sipernat 320 DS (Silica) sold by Degussa AG.

Polyvinylpyrrolidone (PVP) Solution Amount (gm) Buffer Solution 226.03Mega 8¹ 13.23 Geropon T77² 1.405 PVP³ 89.33¹Mega 8 is n-octanoyl-N-methylglucamide sold by Dojindo MolecularTechnologies Inc.²Geropon T77 is N-methyl oleyl taurate sodium salt sold by Rhodia HPCII.³PVP is Polyvinylpyrrolidone K25 sold by BASF.

Trehalose Solution¹ Amount (gm) H₂O 36.4 Trehalose 23.6¹This trehalose solution is used only in preparing the “Enzyme Solution”listed below.

PQQ Solution Amount (gm) Buffer Solution 1^(st) 101.59 addition PQQ0.533 Buffer Solution 2^(nd) 30.0 addition

Amount (gm) Enzyme Solution PQQ Solution 132.12 Gluc-Dor 19.13 (673 U/mgLy) Trehalose Solution 58.75 Mediator Solution Buffer Solution 782.27Mediator 41.26 5 N KOH 36.47

The buffer solution, Keltrol F solution, CMC solution, and the Silicasuspension were prepared a day before. These solutions can then becombined as listed below to prepare the reagent composition. FinalReagent Composition Thickener I 331.51 g (Keltrol F solution) ThickenerII (CMC 1262.9 g Solution) PVP Solution 315.05 g Silica suspension 762.3 g Propiofan solution 257.09 g Mediator Solution 855.84 g EnzymeSolution 196.65 g 5N KOH as required to achieve final pH of 6.9 to 7.1Water (bidistilled) 518.69 gFor this reagent prior to coating, the final pH was 6.96 and did notneed adjustment with 5N KOH solution. The measured viscosity was 111mPas, which is in the correct range for coating of 105 to 115 mPas.

FIGS. 5 and 5A present a flow chart illustrating a preferred process 100for preparing a test strip useful in accordance with the presentinvention. Process 100 begins in the central process line 101 at stage102 with selection of a film material for the base layer or basesubstrate. In a preferred embodiment, the film is provided as acontinuous roll having a width and length suitable for fabricating alarge number of test strips. In subsequent finishing steps, theprocessed film can be subdivided to provide a single strip or web havinga width that approximates the length of the test strip and includes aseries of test strips, or can be die cut to provide individual testsensors.

From stage 102, the film proceeds to stage 104 where it is pretreated toreceive a metal coating and is coated with the metal in one continuousprocess. The pretreatment can be used to clean or modify the surface toprovide a uniform coating thickness and better adhesion of thesubsequent metallized layer. The pretreatment can include subjecting thefilm to corona discharge or Argon plasma. Immediately after thispre-treatment, a uniform conductive coating is applied to the film asshown at 106. Alternatively, suitable substrates with metal coatings canbe obtained commercially.

The metallic layer may contain pure metals, alloys, or other materials,which are metallic conductors. Examples of metals or metallic-likeconductors include: aluminum, carbon (such as graphite), cobalt, copper,gallium, gold, indium, iridium, iron, lead, magnesium, mercury (as anamalgam), nickel, niobium, osmium, palladium, platinum, rhenium,rhodium, selenium, silicon (such as highly doped polycrystallinesilicon), silver, tantalum, tin, titanium, tungsten, uranium, vanadium,zinc, zirconium, mixtures thereof, and alloys or solid solutions ofthese materials. Preferably, the materials are selected to beessentially unreactive to biological systems; such materials include:gold, platinum, palladium, iridium, or alloys of these metals. Themetallic layer may be any desired thickness.

The conductive coating is preferably a metal layer that is applied by avariety of methods, including but not limited to sputtering, physicalvapor deposition (PVD), plasma assisted vapor deposition (PAVD),chemical vapor deposition (CVD), electron beam physical vapor deposition(EBPVD), and/or metal-organic chemical vapor deposition (MOCVD). Vapordeposition is typically performed under vacuum. These techniques arewell known in the art and can be used to selectively provide a uniformlythin coating of metal onto a substrate. The resulting metallized filmcan be inspected to ensure that the metal coating is uniform and free ofmaterial defects.

The roll of metallized film next encounters stage 108 where it issubdivided and/or sized to provide webs having a width that approximatesthe final length of an individual test strip. The slicing can beaccomplished using fixed-knife slitting equipment well-known in the art.

A single web proceeds to stage 110 for patterning the electrodes,traces, and contacts or pads. At this stage, the electrodes, traces, andcontact pads are formed by removing metal from the surface of the webstrip. The excess metal can be removed using a variety of techniqueswell known in the art. At this stage, one or more indexing orregistration marks can be formed either on a first edge proximate to theelectrodes, the opposite second edge proximate to the electrode pad, onboth edges or anywhere in between. The indexing marks, particularlythose at an edge, can be used in subsequent operations to align layeredcomponents prefabricated in separate operations.

In a preferred method, the metal is laser ablated to eliminate undesiredportions of the metal and leave the desired electrical components. Inaccordance with this method, selected areas are laser etchedsimultaneously, in a “broad field”, as opposed to using linear movementof a focused laser beam. This broad field laser ablation method providesa precise metal pattern rapidly and at reduced cost as compared to otherapproaches. Corona treatment of the patterned substrate is thenconducted at stage 111.

The patterned web continues on to stage 112, where a reagent layer isdeposited onto the electrodes. In a preferred embodiment, the reagentlayer is deposited as a continuous elongate stripe extending adjacent orclose to the first edge, and overlying the measuring electrodes formedon the patterned web. As previously noted, the reagent is consequentlylocated across the full width of the test strip, including the arealaterally outside of the sample-receiving chamber and between the basesubstrate and the spacing layer. Also as noted, this will facilitate thedrying of the reagent without discontinuities, edge effects, or othervariances that would detract from providing a thin, flat, uniformreagent layer within the sample-receiving chamber. The reagent includesa combination of components, and is formulated to dry rapidly withminimal or no running after deposition.

This stripe may be applied in any suitable fashion which provides thedesired extent and uniformity of thickness, precision of the stripeedge, homogeneity, and the like. Preferred methods are capable ofapplying the desired coating at relatively high speed and high batchsize. Suitable methods of application are well known in the art andtherefore are not detailed herein.

Preparing the Spacing Layer

Referring now to process line 114, a process flow for preparing thespacing layer is illustrated. Beginning at stage 116, a material isselected to prepare a spacing layer for laminating on top of the reagentcoated web prepared at stage 112. The base film for the substrate can beselected from a variety of materials. The spacing layer material,similar to the base layer, can be provided as an elongate roll which canbe conveniently processed rapidly and with high efficiency. Preferredmaterials include a polyester film sold under the trade name MELINEX® byDuPont. Other materials suitable for use in the present invention couldinclude PEN. The spacing layer material has a thickness specificallyselected to provide a desired chamber depth (or height) in each of thetest strips when combined with the thicknesses of any joining layersthat are used to laminate the spacer to the other strip components. Inpreferred embodiments, the spacing layer is selected to have a thicknessbetween about 75 μm and about 150 μm, more preferably from about 100 μmto about 125 μm. As noted above, the spacing layer can be formed of adouble-sided adhesive.

The spacing layer is preferably formed as a continuous film having aseries of gaps that will align with the electrodes on the bottomsubstrate webbing. The manner of joining the spacing layer and bottomsubstrate will impact the method for preparing the spacing layer. Forexample, if the spacing layer is to be heat welded to the bottomsubstrate, then the spacing layer may simply be die cut to provide theappropriately spaced chamber gaps. However, a preferred method is theuse of thin, non-interfering adhesives that join the adjacent layers. Inaccordance with this preferred method, a spacing layer is prepared forcombination with the previously described substrate webbing as set forthhereafter.

The spacing layer film is prepared having the desired width forcombination with the remainder of the test strip components. The spacinglayer film may include an opaque portion, e.g., a section 23 of it isprinted blue or another color for use in visualizing thesample-receiving chamber, as described elsewhere. The spacing layer filmis laminated on the bottom side with a combination adhesive and releaseliner, and on the top side with a similar combination adhesive andliner.

At stage 118, two transfer adhesives are laminated to the spacing layermaterial: the first transfer adhesive is laminated to the top surface ofthe spacing layer, and the second transfer adhesive is laminated to thebottom surface of the spacing layer. Preferably, the transfer adhesivesare the same adhesive; however, in alternative embodiments, the firstand second transfer adhesives can be different from each other. Inpreferred embodiments, the transfer adhesives are selected from commonlyused and known adhesives, including pressure sensitive adhesives.Preferred adhesives exhibit sufficient hydrophobicity to prevent orinhibit the test sample in the chamber from wicking out between thespacing layer and the reagent layer or base substrate. An example of asuitable sensitive adhesive is ARCare 90132 from Adhesives Research Inc.The adhesives are provided with a release liner to prevent prematureadhesion of the spacing layer during processing. The release liners aredisposed on the exterior surface of the first and second transferadhesives, facing outward from the spacing layer material.

The spacing layer with the adhesive release liners on the top and bottomsurfaces progresses to stage 120. At stage 120, the cavity which willform the sample-receiving chamber is punched in the spacing layer. Inone embodiment, the cavity is punched using a “kiss cut” method. Thekiss cut method cuts through the upper release liner, the upperadhesive, the spacing layer, and the lower adhesive, but not through thelower release liner. In subsequent operations, simply removing the lowerrelease liner will then remove the punched out portions of the loweradhesive, the spacing layer, the upper adhesive, and the upper releaseliner from the punched spacing layer. In other embodiments, the cavitycan be punched through with a hollow die. The hollow die completelypunches or cuts through the spacing layer, the two adhesives, and thetwo release liners, with the punched out portion subsequently removed inthe hollow die. The spacing or pitch between each cavity is determinedand accurately controlled to allow accurate mating of the punchedspacing layer over the electrodes using one or both of the indexingmarks patterned in the reagent-coated web.

At stage 122, the lower release liner on the spacing layer is removed,taking with it the kiss cut portions and exposing the adhesive on theunderside surface of the spacing layer. Proceeding on to stage 124 inprocess line 101, the spacing layer is laminated over the reagent-coatedweb using one or more of the indexing marks previously patterned on theweb to correctly align each cavity formed in the punched spacing layerdirectly on top of an electrode set to provide a web-spacing layerlaminate. At stage 126 in the central process line 101, the upperrelease liner covering the upper adhesive on the web-spacing layerlaminate is removed in preparation for attaching the cover layer.

Laminating on the Cover Portions

At stage 128, a material for a body cover is introduced into theprocess. In preferred examples, the material is a flexible polymericmaterial and may be selected, for example, MELINEX 454 or MELINEX 339from du Pont. The material for the body cover is sized to have a widthsufficient to overlay at least a portion of the electrode traces in thesample-receiving chamber of the test strip.

Referring now to process line 130, beginning at stage 131, a filmmaterial is selected to provide a chamber cover over the cavity,reagent, and measuring electrodes on the web-spacing layer laminate. Inpreferred embodiments, the chamber cover material is provided as a clearpoly(ethylene-terephthalate) (PET) or poly(ethylene-naphthalate) (PEN)film having a thickness between about 100 μm and about 200 μm. Thecoating may preferably include a release liner, which can be removedimmediately prior to laminating over the web-spacing layer. The chambercover is preferably made from a hydrophilic material or the bottomsurface of the chamber cover may be treated or coated to make ithydrophilic as indicated at 134. At stage 138, the film material can besized to a desired width sufficient to form the chamber cover to overlaythe cavity and electrodes.

Proceeding to stage 140, the body cover from stage 128 and the chambercover from stage 138 are laminated to the web-spacing layer laminate. Inpreferred embodiments, the body cover and chamber cover aresimultaneously laminated with the web-spacing layer laminate. The bodycover is positioned over a portion of the electrode traces proximate tothe electrodes formed on the base substrate. The chamber cover ispositioned over the cavity, reagent, and measuring electrodes on theweb-spacing layer laminate. The body cover and chamber cover areseparated by a gap to form a vent at the interior end of the cavityformed in the test strip.

As described, the chamber cover is placed near the edge of the strip tooverlie the cut out portion of the spacing layer, leaving the innermostportion of the cut out uncovered. As just described, this chamber coverpreferably includes a hydrophilic underside to promote the wicking offluid into the reagent chamber. The chamber cover is spaced slightlyfrom the body cover to form a gap which thereby communicates with thesample-receiving chamber and serves as a vent opening for air to escapeas fluid enters the chamber, as described above.

The opacity of the spacing layer and the transparency of the chambercover cooperate to allow a user of the final test strip to better viewthe progress of a test. As constructed, the bottom substrate or reagentlayer coated thereon is visible through the cut out in the spacing layerand through the transparent chamber cover. The bottom substrate and/orreagent has a light color, e.g., bright yellow, which contrasts with theopaque coloring of the spacing layer. Therefore, the progress of a fluidthrough the capillary channel can be easily monitored by the personusing the test. Further, since the slot 34 is configured to behydrophobic on the body cover side and hydrophilic on the chamber coverside, fluid will abruptly stop when it reaches the slot, thus presentinga sharply defined fill-line which in turn provides a clear indication tothe user that sufficient fluid sample has been received into thechamber.

Separating the Test Strips

From stage 140, the finish processing steps for the fabrication of teststrips are performed. At stage 142, a decision is made whether tomanufacture a single, die-cut test strip similar to the single teststrip 10 above discussed. If so, then the multi-layered laminate fromstage 142 proceeds to stage 144 to be die cut into single test strips.

Alternatively, the multi-layered laminate from stage 142 proceeds tostage 148, where it is kiss cut to define individual test strips and toperforate or weaken the boundaries between adjacent test strips on theribbon. Additionally, at stage 149 the ends of the test strips are diecut along the laminated ribbon. One end of the web is cut to form thefluid receiving end of the test sensor with a Y-shaped opening leadinginto the cavity. The test strips may be divided into cards comprising anumber, e.g., 25, of strips which are only fence cut and then folded tobe stacked in a vial or dispenser.

Proceeding out of either stage 144 or 149, the processed strips orribbon of strips are inspected and ultimately packaged for use by theconsumer at stage 146 or 150, respectively.

FIGS. 6-16 illustrate in greater detail some of the components and/orprocess stages previously described with respect to FIGS. 5 and 5A. FIG.6 illustrates a perspective view of one embodiment of a base film foruse in forming the test strip. Base film 160 is preferably provided as aflexible film or web material that is rolled onto one or more rollers162 with processes 164, 166 proceeding on the material between therollers.

The pretreated upper surface of the film is metallized using asputtering, PVD, CVD, EBPVD, MOCVD or another suitable process,illustrated by reference number 166 and described more fully above, todeposit a uniform coating of a metal or metal alloy. The processes canuse a single or multiple target source for the metallic layer. Themetallized film 168 can then be sectioned or subdivided into a pluralityof metallized films, e.g., 170 a, 170 b, and 170 c, by cutting orslicing the film as illustrated by reference number 172. Each separatedroll of the conductive, metallized film 170 can then be rolled upon asingle core or upon a plurality of different cores as preferablydesired.

The electrical components are formed from the conductive film, as shownin one embodiment in FIG. 7. The metallic surface of film 170 is treatedto remove any metallic component that is not desired to form theelectrodes, traces, contact pads, or other intended features. Thisprocess can be precisely controlled using laser ablation or othertechnology. The process provides a plurality of sets of electrodes 182,traces 184, and contact pads 186. The process can also provide aplurality of indexing or registration marks 176 along a first edge 178and/or similar registration marks 177 along the opposite edge 180. Asshown in FIG. 7, repeating features of the electrode pattern formregistration markings 177. Preferably, each set of electrodes and/orcontacts is associated with at least one index or registration mark, 176and 177, respectively.

FIG. 8 illustrates a portion of a reagent-coated web 188. The reagentcomposition is deposited on the surface of the flexible web material.The reagent layer 190 is deposited using a variety of coating methodsincluding curtain coating, hot melt coating, rotary screen coating,doctor blade or air knife coating, Meyer bar coating, and reverse rollcoating techniques. Preferably, the reagent layer is deposited on theflexible web as a wet composition at a thickness of between about 50 μmand about 100 μm, more preferably, between about 60 μm and about 80 μm.Web 188 can be provided by coating a uniformly thin layer of reagent 190directly on top of electrode sets 182 and along the length of web 188 asa continuous narrow band 192. In preferred embodiments, the narrow band192 has a width of between about 5 mm and 9 mm and a dry thickness ofbetween about 2 μm and about 10 μm. As depicted in FIG. 8, the reagentlayer 190 is translucent.

FIG. 9 is an exploded view of a spacing layer assembly 194, which can beassembled in accordance with the present invention. Spacing layerassembly 194 comprises a spacing layer 196 preferably formed of apolymeric material. Spacing layer 196 includes a band or section 197that is colored (corresponding to section 23, FIG. 2). In themanufacturing process, spacing layer 196 is provided in a roll 198 andis then overcoated with adhesives on top and bottom.

The top adhesive is provided in a roll 200 which further comprises a topor “tight” release liner 202, which is adapted to withstand furtherprocessing, an adhesive 204, and a lower or “easy” release liner 206.Preferred adhesives 204 for use in the present invention include apressure-sensitive adhesive sold under the trade name ARCare 90132 byAdhesives Research Inc. During assembly, bottom release liner 206 isremoved and the resulting adhesive 208 having the top release liner 202still present is adhered to spacing layer 196 as indicated in the top ofFIG. 9.

Similarly, the bottom adhesive is provided in a roll 210 which furthercomprises a top or “tight” release liner 212, which is adapted towithstand further processing, an adhesive 214, and a lower or “easy”release liner 216. Preferred adhesives 214 for use in the presentinvention include a pressure-sensitive adhesive sold under the tradename ARCare 90132 by Adhesives Research Inc. During assembly, bottomrelease liner 216 is removed and the resulting adhesive 218 having itstop release liner 212 facing away from spacing layer 196 is adhered tospacing layer 196 as indicated in FIG. 9. It should be understood thatadhesive 204 can be the same or different from adhesive 214.

FIG. 10 illustrates spacing layer 196 that has been die cut to formpre-capillaries 220 a, 220 b, 220 c, etc., and is ready to be laminatedto a web of base substrate material 188 as described with reference toFIG. 8. Pre-capillaries 220 can be formed using a “kiss-cut” techniquein which a die cuts through the top release liner 202, adhesive 204,spacing layer 196, and adhesive 214, but not release liner 212, which,as noted above, faces away from spacing layer 196. Release liner 212 isthen removed along with portions of the top release liner 202, adhesive204, spacing layer 196, and adhesive 214 that had been cut through.These portions that are cut through comprise “capillary trim,” i.e., asandwich of layers shaped like pre-capillaries 220. This “trim” isremoved along with release liner 212, leaving the cavities 220 devoid ofany material. As release liner 212 is removed, it can be inspected toensure that it always contains the capillary trim just described. Theresulting series of cavities 220 are spaced from each other a desireddistance selected to position each one of the channels of the series ofchannels 220 directly over a measuring electrode set in the test strip.The spacing layer 196 having its lower adhesive exposed can then bealigned with web 188 by means of indexing marks 176 and laminatedthereto. Each capillary channel of the series of channels 220 overlaysone set of measuring electrodes 182.

FIG. 11 illustrates an assembly 230 formed by the lamination of spacinglayer 196 to web 188. In FIG. 11, the upper release liner 202 has beenremoved from the top adhesive 208, which makes assembly 230 ready forassembly of additional material thereto. As shown in FIG. 12, a web 240of chamber covering layer material and a web 234 of body coveringmaterial are aligned over the exposed upper adhesive 208 of assembly 230and are ready to be adhered thereto. As depicted in FIG. 12, chambercovering layer 240 is clear and includes a hydrophilic coating (seecoating 21, FIG. 2) on at least the side that faces cavities 220. Thisfacilitates wicking or transport of the liquid sample into thesample-receiving chamber and over the electrodes and reagent layer. Bodycover 234 is opaque, is colored as shown, and is preferably hydrophobic.Covering layer 240 and body cover 234 can be provided on reels likethose described above with reference to FIG. 9.

Preferably, chamber covering material 240 is slightly thinner than bodycovering material 234. After the chamber covering material 240 and bodycovering material 234 are laminated to the other layers (describedbelow), the assembly is rewound to await the final processing steps. Ifbody covering material 234 is thicker than chamber covering material240, then body covering material 234 will absorb more of the pressure orforce imparted to the web as it is rewound and stored. Thus, if anyadhesive squeezes out of the web as it is rewound, the adhesive willsqueeze out around the body covering material 234 and not the chambercovering material 240. Advantageously, the thinner chamber cover thusreduces the possibility of the adhesive squeezing out from under itduring roll processing and entering the capillary zone where it coulddegrade or destroy the test strips ultimately produced.

Assembly 260 shown in FIG. 13 is produced by laminating webs 234 and 240to the assembly 230 shown in FIG. 12 and then trimming the end of theweb to form dosing edge 250. Dosing edge 250 is preferably formed by ashear cut in which the cutting blade moves across the end of the web asindicated by arrow 252. By contrast, it is more difficult to use a diepunching technique without damaging the capillaries. The shear cut alongdosing edge 250 also cuts away a portion of the pre-capillaries 220 anddefines the final volume of capillaries 222. Capillaries 222 preferablyinclude a flared or Y-shaped opening as shown. Preferably, a gap 262 isformed between the chamber covering web and the body covering web andthis gap will ultimately provide a vent opening in the individual teststrips. In preferred embodiments, the gap has a width of between 1.0 mmand about 1.6 mm. As noted above, however, the gap could be replaced byusing a unitary covering layer having a notch formed on its underside(FIG. 1B) or by having the chamber cover overlap the body cover or viceversa. (FIG. 1C).

With further reference to FIG. 13, assembly 260 is ready for furtherprocessing as indicated by dashed lines 262 and 264. In FIG. 14 there isshown the kiss-cut strip 276 having a plurality of individual teststrips, e.g., 278 a, 278 b, and 278 c, detachably connected together. Itcan be observed that kiss-cut strip 276 has been trimmed or cut at itsupper end along lines 262 in FIG. 13 to have a profile and/orconfiguration suitable to facilitate capturing a very small fluid samplein each of the series of capillary channels 222. In the illustratedembodiment, kiss-cut strip 276 has a flat working end 280 exposing theend of the sets of Y-cut capillary channels 222. The resultingconfiguration of second edge 282 can be provided to facilitate insertionof a single strip into a meter (not shown). For example, the second edge282 can have a registration mark and/or tabs, cut slots, or otherconfigurations designed to allow insertion of a single strip into themeter in only one direction (see arrow 31, FIG. 1)

With reference to FIG. 13, the edges 177 of contact pads 288 are spacedby a constant pitch, “P” as shown, and edges 177 can therefore be usedas registration marks. As in other processing steps, the indexing orregistration marks 176 and 177 on either the first edge and/or thesecond edge can be used to accurately “kiss cut” and trim the individualtest strips from the laminated structure 260.

FIG. 15 is a perspective view of one embodiment of a punched cut teststrip 290 formed by cutting through the dashed lines 264 shown in FIGS.13 and 14. Strip 290 illustrated in FIG. 15 has been substantiallydescribed above as test strip 10. Strip 290 is provided as an individualtest strip separate from any other test strip.

EXAMPLE

By way of specific example, a test strip is formed based on thedescribed method and using materials as follows. The bottom substrate issurface coated with a 50 nm layer of gold, and is slit to widths of43-45 mm. Laser ablation (308 nm) is performed using a field size ofapproximately 40 mm×10 mm. The spacing layer assembly includes a spacinglayer film of white Melinex™ 339, and a thickness of 0.1016 or 0.127 mm(4 or 5 mil). The bottom and top adhesives are an Adhesive ResearchArcare 90132 adhesive at 0.0254 or 0.0127 mm (1 or 2 mil), sandwichedbetween release liners having a thickness of 0.0508 mm (2 mil). Thecapillary channels are formed with a width of 1.500 mm, +/−0.050 mm, anda pitch (spacing) of 9 mm, +/−0.150 mm.

The body cover 18 comprises a strip of Melinex 454, 453 or 339 material,0.127 mm (5 mil) thick. The chamber cover 20 comprises a polyester orpolyethylene naphthate material formed, for example, from Melinex 454 or453, 0.1016 mm (4 mil) thick. As indicated, the chamber cover may bepreferably treated or coated to have a hydrophilic underside adjacent tothe capillary channel to promote wicking of the blood specimen into thechannel. In a preferred embodiment, a Melinex 453 foil (4 mil) forchamber cover 20 is coated on its underside with a hydrophilic material21, ARCare 90037 from Adhesives Research Inc. Preferably the chambercover material is initially formed as a wider material, and is slit tothe desired width after preparation.

Test Strip Examples

The following materials will be used in the strip: Base substrate layer12 Melinex 329-9 mil or 329 - 10 mil Conductive Layer 26 Sputteredgold - 50 nm Lower Adhesive Layer 49 AR ARCare 90132 PSA - 1 to 0.5 milSpacing layer 14 Melinex 329 or 339 - 4 to 5 mil Adhesive Layer 46 ARARCare 90132 PSA - 1 to 0.5 mil Body Cover 18 Melinex 339 or 329 or454 - 5 mil Chamber Cover 20 Melinex 339 or 329 or 454 - 4 milHydrophilic foil 21 ARCare 90037Storage of Strips

Strips may be packaged in a variety of ways. For example, strips may bepackaged into flip-top plastic vials (e.g., 10, 25 or 50 count). Allcontainers include desiccant materials necessary to ensure acceptableshelf-life. Test strips preferably display a minimum shelf life of 18months when stored between 4°-32° C. in tightly closed containers asprovided.

While preferred embodiments incorporating the principles of the presentinvention have been disclosed hereinabove, the present invention is notlimited to the disclosed embodiments. Instead, this application isintended to cover such departures from the present disclosure as comewithin known or customary practice in the art to which this inventionpertains and which fall within the limits of the appended claims.

1. A test strip, comprising: a base substrate having a reagent layerdisposed thereon; a covering layer overlying the base substrate andcomprising a chamber cover and a body cover having a slot therebetween,the slot being positioned over the reagent layer; and a sample receivingchamber disposed between the base substrate and the covering layer, theslot being in communication with the sample receiving chamber anddefining a vent opening in the covering layer that allows air to escapeas fluid enters the sample receiving chamber.
 2. The test strip of claim1, wherein the slot comprises a gap which spaces the body cover from thechamber cover.
 3. The test strip of claim 2, wherein the gap extendsacross the width of the covering layer.
 4. The test strip of claim 1,wherein the covering layer is unitary and the slot comprises a recessformed on the underside thereof, the recess extending to a side of thetest strip and terminating at the vent opening.
 5. The test strip ofclaim 4, wherein the recess extends across the width of the test stripand the vent opening comprises two vent openings disposed on oppositesides of the test strip.
 6. The test strip of claim 1, wherein the slotis substantially straight.
 7. The test strip of claim 6, wherein theslot comprises a fill line.
 8. The test strip of claim 6, wherein theslot is oriented substantially perpendicular to a longitudinal axis ofthe test strip.
 9. The test strip of claim 1, wherein the chamber coverand the body cover are disposed in substantially the same horizontalplane.
 10. The test strip of claim 9, wherein the chamber cover and thebody cover are positioned end to end in a lengthwise direction along thetest strip.
 11. The test strip of claim 1, further comprising anelectrode disposed in the sample receiving chamber, the electrode beingcovered by the reagent layer.
 12. The test strip of claim 1, wherein thechamber cover overlies the sample-receiving chamber.
 13. The test stripof claim 12, wherein the slot comprises a hydrophobic side adjacent thebody cover, further wherein sample fluid entering the sample receivingchamber will progress to the slot but no farther, whereby the slotcomprises a fill line.
 14. The test strip of claim 13, wherein thechamber cover is transparent or translucent, whereby progression ofsample fluid into the sample receiving chamber to the fill line isvisible.
 15. The test strip of claim 13, wherein the chamber covercomprises a hydrophilic surface on an underside thereof, whereby wickinginto the sample-receiving chamber to the slot is promoted.
 16. The teststrip of claim 13, wherein the slot comprises a gap that spaces thechamber cover from the body cover.
 17. The test strip of claim 1,wherein the body cover is adhered to the test strip with a hydrophobicadhesive.
 18. The test strip of claim 1, wherein the underside of thechamber cover is hydrophilic.
 19. The test strip of claim 18, whereinthe underside of the chamber cover is coated with a hydrophilicsubstance.
 20. The test strip of claim 1, wherein the chamber cover andbody cover are formed from the same material.
 21. The test strip ofclaim 1, further comprising: electrodes disposed in the sample receivingchamber; contact pads formed on the base substrate at a meter insertionend of the test strip; and electrode traces extending along the basesubstrate and connecting the electrodes to the contact pads, thecovering layer covering the electrode traces and exposing the contactpads.
 22. The test strip of claim 1, further comprising a workingelectrode and a counter electrode disposed within the sample receivingchamber, the slot being positioned downstream of the working and counterelectrodes.
 23. The test strip of claim 1, wherein the chamber coveroverlies the sample receiving chamber and the chamber cover istransparent or translucent.
 24. The test strip of claim 1, wherein theslot is substantially aligned with an interior end of the samplereceiving chamber.
 25. The test strip of claim 1, further comprising aspacing layer disposed between the covering layer and the basesubstrate, the spacing layer defining a void portion that furtherdefines the height and perimeter of the sample receiving chamber betweenthe base and the covering layer.
 26. The test strip of claim 25, whereinthe void portion forms a channel that begins at an opening at an edge ofthe test strip, the opening being in communication with the samplereceiving chamber.
 27. The test strip of claim 26, wherein the voidportion terminates at a position substantially aligned with the slot.28. A test strip, comprising: a base substrate having electrodes formedthereon; a covering layer overlying the base substrate, the coveringlayer comprising a chamber cover and a body cover having a slottherebetween; and a unitary spacing layer disposed between the coveringlayer and the base substrate, the spacing layer having a void thatdefines a sample receiving chamber between the base substrate and thecovering layer; wherein the slot is in communication with the samplereceiving chamber, thereby defining a vent opening in the covering layerthat allows air to escape as fluid enters the sample receiving chamber.29. The test strip of claim 28, wherein the slot is substantiallyaligned with an interior end of the sample receiving chamber.
 30. Thetest strip of claim 28, wherein one surface of the spacing layer isadhered to the base substrate and an opposite surface of the spacinglayer is adhered to the covering layer.
 31. The test strip of claim 28,wherein the chamber cover comprises a hydrophilic surface adjacent thesample receiving chamber.
 32. The test strip of claim 28, furthercomprising a reagent layer disposed in the sample receiving chambercovering at least one of the electrodes.
 33. The test strip of claim 32,wherein the reagent layer extends under the spacing layer and issandwiched between the spacing layer and the base substrate.
 34. Thetest strip of claim 33, wherein the reagent layer extends across thewidth of the test strip.
 35. The test strip of claim 32, wherein thereagent layer substantially covers the bottom of the sample receivingchamber.
 36. The test strip of claim 32, wherein the slot is positionedover the reagent layer.
 37. The test strip of claim 28, wherein thesample receiving chamber is bounded on the top side thereof by thechamber cover and the underside of the chamber cover is hydrophilic,whereby wicking of sample fluid into the sample receiving chamber ispromoted.
 38. The test strip of claim 28, wherein the slot comprises agap that extends across the covering layer and separates the body coverfrom the chamber cover, the body cover and the chamber cover havingsubstantially straight edges proximate the gap.
 39. The test strip ofclaim 38, wherein the gap is oriented substantially perpendicular to alongitudinal axis of the test strip.
 40. The test strip of claim 28,wherein the sample receiving chamber communicates with a fluid receivingopening disposed at an edge of the test strip.
 41. The test strip ofclaim 28, wherein the body cover includes a hydrophobic portion adjacentthe slot.
 42. The test strip of claim 28, wherein the body cover isadhered to the spacing layer with a hydrophobic adhesive.
 43. The teststrip of claim 28, wherein the body cover and chamber cover are formedfrom the same material.
 44. The test strip of claim 43, wherein the bodycover is treated with a hydrophobic substance and the chamber cover istreated with a hydrophilic substance.
 45. The test strip of claim 28,further comprising electrode traces extending from the electrodes alongthe base substrate and terminating in contact pads at a meter insertionend of the test strip, the contact pads being exposed for electricalconnection thereto.
 46. The test strip of claim 28, wherein the chambercover overlies substantially the entire length of the sample receivingchamber.
 47. The test strip of claim 46, wherein the chamber cover istransparent or translucent above the sample receiving chamber, wherebyfluid entering the sample receiving chamber is visible through thechamber cover.
 48. The test strip of claim 47, wherein a top surface ofthe spacing layer adjacent the void and the bottom of the samplereceiving chamber have contrasting colors and are visible through thechamber cover.
 49. The test strip of claim 28, wherein the basesubstrate, spacing layer and covering layer are adhered to one anotherby one of heat welding, laser welding, heat sensitive adhesives,pressure sensitive adhesives and combinations thereof.
 50. The teststrip of claim 28, wherein the electrodes are disposed in the samplereceiving chamber.
 51. The test strip of claim 50, further comprisingcontact pads disposed at a meter insertion end of the test strip andelectrode traces extending along the base substrate connecting theelectrodes to the contact pads.
 52. The test strip of claim 51, whereinthe covering layer and spacing layer are sized to expose the contactpads, whereby the contact pads can be electrically connected to a meter.53. The test strip of claim 52, wherein the chamber cover overliessubstantially the entire length of the sample receiving chamber and hasabout the same length as the sample receiving chamber.
 54. The teststrip of claim 53, wherein the chamber cover is transparent ortranslucent.
 55. The test strip of claim 28, wherein the body cover andchamber cover are aligned end to end and are disposed in substantiallythe same horizontal plane.
 56. A method of manufacturing a plurality oftest strips, said method comprising: (a) providing a web of basesubstrate material; (b) forming a plurality of electrode sets on theweb; (c) providing a reagent layer covering at least one electrode ofeach electrode set; then (d) providing a continuous web of spacingmaterial having a series of cavities formed therein and laminating theweb of spacing material over the web of base substrate material suchthat each one of the cavities aligns with a respective one of theelectrode sets; (e) providing a web of covering layer made from twopieces and laminating it over the web of spacing material such that thetwo pieces are separated by a gap and the gap is positioned over theseries of cavities; and (f) cutting the web into the plurality of teststrips.
 57. The method of claim 56, wherein the reagent layer is appliedin a uniform thickness as continuous stripe along the web of basesubstrate material.
 58. The method of claim 57, wherein step (c) furthercomprises depositing the reagent stripe over the plurality of electrodesets.
 59. The method of claim 56, wherein step (b) comprises applying aconductive coating to the web of base substrate and then removingportions of the conductive coating to form the electrode sets.
 60. Themethod of claim 59, wherein the portions of the conductive coating areremoved by laser ablation.
 61. The method of claim 60, wherein the laserablation comprises broad field laser ablation.
 62. The method of claim56, wherein the two pieces have substantially straight edges, wherebythe gap is substantially straight and extends over the series ofcavities.
 63. The method of claim 56, wherein each of the two pieces ofcovering material comprises an elongated continuous web.
 64. The methodof claim 63, further comprising making a series of substantiallyequidistant cuts between the electrode sets, the cuts beingsubstantially perpendicular to the gap, whereby each individual teststrip formed thereby has a gap extending across its covering layer, theindividual gaps forming vent openings communicating with respectivecavities.
 65. The method of claim 64, wherein the cutting furthercomprises cutting one end of the web to form fluid receiving ends of thetest strips with openings at their edges, each opening leading into arespective cavity.
 66. The method of claim 65, wherein the fluidreceiving ends are Y-shaped.
 67. The method of claim 65, wherein thecavities extend from the openings at the edges of the test strips to thevent openings formed in the covering layers.
 68. The method of claim 56,further comprising positioning the gap over an interior end of theseries of cavities.
 69. The method of claim 56, further comprisingapplying the two pieces to the web at substantially the same time. 70.The method of claim 56, further comprising providing one piece of thecovering layer web with a hydrophilic surface and the other piece ofcovering layer web with a hydrophobic surface.
 71. A test strip,comprising: a test strip body; a sample receiving chamber disposed inthe test strip body; a fluid receiving opening disposed at an edge ofthe test strip body and communicating with the sample receiving chamber;a covering layer at least partially defining the top of the test stripbody, the covering layer comprising two pieces having a slottherebetween, the slot forming a vent opening positioned over and incommunication with the sample receiving chamber, whereby air can escapethrough the vent opening as fluid enters the sample receiving chamber;and the slot comprising a hydrophobic edge, wherein sample fluid canprogress into the sample-receiving chamber to the slot, but no farther,whereby the slot comprises a fill line.
 72. The test strip of claim 71,further comprising a reagent layer and at least one electrode disposedin the sample receiving chamber.
 73. The test strip of claim 72, whereinthe slot is positioned over the reagent layer.
 74. The test strip ofclaim 71, further comprising a base substrate defining the bottom of thetest strip body, the base substrate having electrodes formed thereon.75. The test strip of claim 74, further comprising a reagent layercovering at least one of the electrodes, the at least one electrodebeing located within the sample receiving chamber.
 76. The test strip ofclaim 71, wherein the test strip body further comprises: a basesubstrate layer defining the bottom of the test strip and havingelectrodes formed thereon; a spacing layer disposed between the coveringlayer and the base substrate layer; and the spacing layer having a voidthat partially defines the boundaries of the sample receiving chamber,the void originating at the fluid receiving opening and terminating at alocation substantially aligned with the slot.
 77. The test strip ofclaim 71, wherein the sample receiving chamber extends inwardly from thefluid receiving opening and terminates at a location substantiallyaligned with the slot.
 78. The test strip of claim 77, wherein a firstof the two pieces of the covering layer overlies the sample receivingchamber, the first piece having an end thereof substantially alignedwith the fluid receiving opening.
 79. The test strip of claim 78,wherein the test strip body has an elongate shape and the samplereceiving chamber extends lengthwise with respect to the test stripbody, the fluid receiving opening being disposed at an end of the teststrip body.
 80. The test strip of claim 71, wherein the two pieces ofthe covering layer are formed of the same material, one of the piecesbeing treated with a hydrophobic substance and the other being treatedwith a hydrophilic substance.
 81. The test strip of claim 80, whereinthe piece of the covering layer treated with the hydrophilic substanceoverlies the sample receiving chamber.
 82. The test strip of claim 71,further comprising a hydrophilic surface in the sample receiving chamberto promote wicking of the sample into the sample receiving chamber. 83.The test strip of claim 82, wherein the hydrophilic surface comprisesthe bottom of the first piece of the covering layer.
 84. The test stripof claim 71, wherein the two pieces of the covering layer are disposedend to end in a lengthwise direction along the test strip.
 85. The teststrip of claim 84, wherein the two pieces of the covering layer aredisposed in substantially the same plane.
 86. A method of testing afluid sample for the presence or concentration of an analyte,comprising: (a) providing a test strip having a sample-receiving openingat an edge thereof, the sample-receiving opening leading into asample-receiving chamber that extends into the test strip; (b) providinga covering layer for the test strip formed with a slot that overlies thesample receiving chamber; (c) placing a fluid sample at the samplereceiving opening; (d) drawing the sample into the sample receivingchamber by capillary action while at the same time displacing air fromthe sample receiving chamber through the slot; (e) continuing to drawthe sample into the sample receiving chamber until the sample reachesthe slot and then halting further progress of the sample beyond theslot, whereby the slot comprises a fill line; and (f) determining thepresence or concentration of the analyte.
 87. The method of claim 86,wherein the covering layer has a transparent or translucent portion thatoverlies the sample receiving chamber such that the sample receivingchamber is visible through the covering layer, the method furthercomprising observing the progression of the sample from thesample-receiving opening to the slot.
 88. The method of claim 86,wherein the halting of further progress of the sample beyond the slotcomprises providing the slot with a hydrophobic edge.
 89. The method ofclaim 86, further comprising providing a reagent layer in the samplereceiving chamber and positioning the slot over the reagent layer. 90.The method of claim 86, wherein the slot comprises a gap, wherein atleast a portion of the air displaced exits from the top of the teststrip.
 91. The method of claim 86, wherein the chamber cover and bodycover are disposed in substantially the same plane.
 92. The method ofclaim 91, wherein the chamber cover and body cover are arranged end toend.
 93. A method of testing a fluid sample for the presence orconcentration of an analyte, comprising: (a) providing a test striphaving a sample-receiving opening at an edge thereof, thesample-receiving opening leading into a sample-receiving chamber thatextends inwardly on the test strip; (b) providing a covering layer forthe test strip comprising a chamber cover and a body cover with a gaptherebetween, the gap overlying a portion of the sample receivingchamber; (c) placing a fluid sample at the sample receiving opening; (d)drawing the sample into the sample receiving chamber by capillary actionwhile at the same time displacing air from the sample receiving chamberthrough the gap, wherein at least a portion of the air displaced exitsfrom the top of the test strip; and (e) determining the presence orconcentration of the analyte.
 94. The test strip of claim 93, whereinstep (d) further comprises drawing the sample into the sample receivingchamber until the sample reaches the gap and then halting furtherprogress of the sample beyond the gap, wherein the gap comprises a fillline.
 95. The method of claim 94, wherein the covering layer has atransparent or translucent portion that overlies the sample receivingchamber such that the sample receiving chamber is visible through thecovering layer, the method further comprising observing the progressionof the sample from the sample-receiving opening to the gap.
 96. Themethod of claim 93, wherein the chamber cover and body cover aredisposed in substantially the same plane.
 97. The method of claim 96,wherein the chamber cover and body cover are arranged end to end.
 98. Atest strip, comprising: a base substrate having a reagent layer disposedthereon; a covering layer overlying the base substrate and comprising achamber cover and a body cover having a slot therebetween, the slotbeing formed as one of a notch on the underside of the covering layerand an overlap between the covering layer and the chamber cover; and asample receiving chamber disposed between the base substrate and thecovering layer, the slot being in communication with the samplereceiving chamber and defining a vent opening in the covering layer thatallows air to escape as fluid enters the sample receiving chamber. 99.The test strip of claim 98, wherein the slot is positioned over thereagent layer.
 100. The test strip of claim 98, wherein the slot extendsacross the width of the covering layer.
 101. The test strip of claim 98,wherein the covering layer is unitary and the slot comprises the notch.102. The test strip of claim 98, wherein the slot comprises the overlapbetween the chamber cover and the body cover.
 103. The test strip ofclaim 102, wherein the overlap comprises the chamber cover covering anedge of the body cover.
 104. The test strip of claim 102, wherein theslot extends across the width of the test strip and the vent openingcomprises two vent openings disposed on opposite sides of the teststrip.
 105. The test strip of claim 98, wherein the slot issubstantially straight.
 106. The test strip of claim 105, wherein theslot comprises a fill line.
 107. The test strip of claim 98, wherein theslot is oriented substantially perpendicular to a longitudinal axis ofthe test strip.
 108. A test strip, comprising: a base substrate having areagent layer disposed thereon; a covering layer overlying the basesubstrate and comprising a chamber cover and a body cover having a slottherebetween, the body cover being thicker than the chamber cover; and asample receiving chamber disposed between the base substrate and thecovering layer, the slot being in communication with the samplereceiving chamber and defining a vent opening in the covering layer thatallows air to escape as fluid enters the sample receiving chamber. 109.The test strip of claim 108, wherein the slot comprises a gap whichspaces the body cover from the chamber cover.
 110. The test strip ofclaim 109, wherein the gap extends across the width of the coveringlayer.
 111. The test strip of claim 108, wherein the slot issubstantially straight.
 112. The test strip of claim 111, wherein theslot comprises a fill line.
 113. The test strip of claim 111, whereinthe slot is oriented substantially perpendicular to a longitudinal axisof the test strip.
 114. The test strip of claim 108, wherein the chambercover and the body cover are disposed in substantially the samehorizontal plane.
 115. The test strip of claim 114, wherein the chambercover and the body cover are positioned end to end in a lengthwisedirection along the test strip.
 116. The test strip of claim 108,wherein the chamber cover overlies the sample receiving chamber. 117.The test strip of claim 116, wherein the slot comprises a hydrophobicside adjacent the body cover, further wherein sample fluid entering thesample receiving chamber will progress to the slot but no farther,whereby the slot comprises a fill line.
 118. The test strip of claim116, wherein the chamber cover is transparent or translucent, wherebyprogression of sample fluid into the sample receiving chamber to thefill line is visible.
 119. The test strip of claim 117, wherein thechamber cover comprises a hydrophilic surface on an underside thereof,whereby wicking into the sample-receiving chamber to the slot ispromoted.
 120. The test strip of claim 117, wherein the slot comprises agap that spaces the chamber cover from the body cover.
 121. The teststrip of claim 108, wherein the chamber cover and body cover are formedfrom the same material.
 122. A method of manufacturing test strips, saidmethod comprising: (a) providing a first web of material having a seriesof spaced cavities formed therein, the cavities defining walls and afloor of a sample receiving chamber and the cavities being open on theirtops; (b) covering the first web of material with a second web ofmaterial, the second web having a first portion having a first thicknessand a second portion having a second thickness greater than the firstthickness, wherein the first portion is aligned over and covers thecavities; and (c) cutting the structure produced in step (b) into thetest strips.
 123. The method of claim 122, further comprising laminatingthe second web over the first web such that the first portion and thesecond portion are separated by a gap.
 124. The method of claim 123,further comprising positioning the gap over the series of cavities. 125.The method of claim 123, wherein the first portion and the secondportion each have a substantially straight edge, whereby the gap issubstantially straight and extends over the series of cavities.
 126. Themethod of claim 122, further comprising forming a plurality of electrodesets on the first web of material.
 127. The method of claim 126, furthercomprising aligning each one of the cavities with a respective one ofthe electrode sets.
 128. The method of claim 126, further comprisingproviding a reagent layer covering at least one electrode of theelectrode sets.
 129. The method of claim 128, wherein the reagent layeris applied in a uniform thickness as a continuous stripe along the firstweb of material.
 130. The method of claim 129, further comprisingdepositing the reagent stripe over the plurality of electrode sets. 131.The method of claim 122, wherein the second web comprises two layers ofmaterial that are separated by a gap that overlies the cavities.